Method of Modulating Cell Resistance

ABSTRACT

The present invention relates to the use of one or more cas genes for modulating resistance in a cell against a target nucleic acid or a transcription product thereof.

FIELD OF INVENTION

The present invention relates to inter alia modulating the resistance ofa cell against a target nucleic acid or a transcription product thereof.In particular, the present invention relates, in one aspect, to the useof one or more cas genes or proteins for modulating the resistance of acell against a target nucleic acid or a transcription product thereof.

BACKGROUND TO THE INVENTION

Cultures—such as starter cultures—are used extensively in the foodindustry in the manufacture of fermented products including milkproducts (such as yoghurt, butter and cheese), meat products, bakeryproducts, wine and vegetable products. The preparation of cultures islabour intensive, occupying much space and equipment, and there is aconsiderable risk of contamination with spoilage bacteria and/or phagesduring the step of propagation. The failure of bacterial cultures bybacteriophage (phage) infection and multiplication is a major problemwith the industrial use of bacterial cultures. There are many differenttypes of phages with varying mechanisms to attack bacteria. Moreover,new strains of bacteriophages appear.

Strategies used in industry to minimise bacteriophage infection, andthus failure of a bacterial culture, include the use of: (i) mixedstarter cultures; and (ii) the alternate use of strains having differentphage susceptibility profiles (strain rotation).

(i) Traditionally, starter cultures in the dairy industry are mixturesof lactic acid bacterial strains. The complex composition of mixedstarter cultures ensures that a certain level of resistance to phageattack is present. However, repeated sub-culturing of mixed straincultures leads to unpredictable changes in the distribution ofindividual strains and eventually undesired strain dominance. This inturn may lead to increased susceptibility to phage attack and risk offermentation failures.

(ii) The rotation of selected bacterial strains which are sensitive todifferent phages is another approach to limit phage development.However, it is difficult and cumbersome to identify and select asufficient number of strains having different phage type profiles toprovide an efficient and reliable rotation program. In addition, thecontinuous use of strains requires careful monitoring for new infectiousphages and the need to quickly substitute a strain which is infected bythe new bacteriophage by a resistant strain. In manufacturing plantswhere large quantities of bulk starter cultures are made ahead of time,such a quick response is usually not possible.

Several attempts have been made to improve the resistance of culturesfor use in industry.

Pedersen et at (7^(th) symposium on lactic acid bacteria: genetics,metabolism and applications, Sep. 1-5, 2002, Egmond aan Zee, TheNetherlands) teach a phage resistant Lactococcus lactis strain, whichhas no thymidylate synthase activity and which requires thymidine forDNA replication.

WO 01/14520 discloses a lactic acid bacterium which have a reducedsusceptibility towards attack by at least one type of bacteriophage.Said lactic acid bacteria comprise a mutated gene involved in pyrimidinemetabolism, namely pyrG which results in a defect in CTP-synthetase.

Kosuge et al (1998—Appl. Environ. Microbiol., Volume: 64, Issue: 11,Page(s): 4328-4332) and Kosuge et al (1994—FEMS Microbiology Letters,123 (1/2) 55-62) teach a Thermus thermophilus HB27 bacterium which ismutated in the proB gene and is unable to utilise proline for growth.

However, there is a continuing need to improve cultures for use inindustry.

SUMMARY OF THE INVENTION

There is described herein the use of CRISPR loci or a component thereoffor modulating the resistance of a cell against a target nucleic acid ora transcription product thereof.

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats)(also known as SPIDRs—SPacer Interspersed Direct Repeats) constitute afamily of recently described DNA loci. CRISPR loci consist of short andhighly conserved DNA repeats (typically 24 to 40 bp, repeated from 1 to140 times) which are partially palindromic. The repeated sequences(usually specific to a species) are interspaced by variable sequences ofconstant length (typically 20 to 58 bp depending on the CRISPR). Up to20 distinct CRISPR loci have been found within a single chromosome.

Although the biological function of CRISPR loci is unknown somehypotheses have been proposed. For example, it has been proposed thatthey may be involved in the attachment of the chromosome to a cellularstructure, or in the chromosome replication and replicon partitioning(Jansen et al., 2002; Pourcel et al., 2005). Moreover, Mojica et al.2005 hypothesis that CRISPR could be involved in conferring specificimmunity against foreign DNA and Pourcel et al. (2005) hypothesise thatCRISPRs are structures that are able to take up pieces of foreign DNA aspart of a defence mechanism. Bolotin et al. (2005) suggest that theCRISPR spacer elements are the traces of past invasions byextrachromosomal elements, and hypothesise that they provide a cell withimmunity against phage infection, and more generally foreign DNAexpression, by coding an anti-sense RNA. Bolotin et al. (2005) alsosuggest that cas genes are necessary for CRISPR formation.

In contrast to the teachings of the prior art which hypothesise thatCRISPR or CRISPR spacers could be involved in conferring specificimmunity, the present invention is based, in part, on the surprisingfinding that cas genes or proteins are required for immunity against atarget nucleic acid or a transcription product thereof.

Even more surprisingly, the inventors have discovered that one or morecas genes or proteins are associated with two or more CRISPR repeatswithin CRISPR loci. In other words, cas genes or proteins seem to bespecific for a given DNA CRISPR repeat, meaning that cas genes orproteins and the repeated sequence form a functional pair. Accordingly,one or more CRISPR spacers may be used together with one or more ofthese functional pairs (i.e. CRISPR repeats and cas genes) in order tomodulate the resistance of a cell against a target nucleic acid or atranscription product thereof.

In one embodiment, for one or more CRISPR spacers to confer immunity tothe cell, the CRISPR repeat(s) and the cas gene(s) or proteins form afunctional combination ie. the CRISPR repeat(s) and the cas gene(s) orproteins are compatible.

Accordingly, we suggest here for the first time that a cas gene orprotein influences resistance—such as the resistance of a bacteria toone or more bacteriophages. In particular, the knowledge of two or moreCRISPR repeats and/or one or more cas genes or proteins for a given cellwill be an advantage to predict, determine and modify its resistance,for example, its lysotype, which defines the resistance/sensitivity of agiven bacterium to various bacteriophages. Consequently, identificationand detection of CRISPR loci in, for example, cells and bacteriophagescould help to determine, predict and modify the resistance profile of acell or phage-host interactions.

Advantageously, the application of one or more CRISPR loci, two or moreCRISPR repeats, one or more cas genes or proteins and/or one or moreCRISPR spacers in genetic engineering could lead to resistant orsensitive variants of cells for use within a wide variety ofapplications in the biotechnology industry.

SUMMARY ASPECTS OF THE PRESENT INVENTION

In one aspect there is provided the use of one or more cas genes orproteins for modulating resistance in a cell against a target nucleicacid or a transcription product thereof.

In a second aspect there is provided the use of a recombinant nucleicacid sequence comprising at least one cas gene and at least two CRISPRrepeats together with at least one CRISPR spacer, wherein at least oneCRISPR spacer is heterologous to at least one cas gene and/or at leasttwo CRISPR repeats to modulate resistance against a target nucleic acidor transcription product thereof.

In a third aspect there is provided a nucleic acid sequence consistingessentially of at least one cas gene.

In a fourth aspect there is provided a nucleic acid sequence consistingessentially of at least one cas gene and at least two CRISPR repeats.

In a fifth aspect there is provided a nucleic acid sequence consistingessentially of at least one cas gene and at least one CRISPR spacer.

In a sixth aspect there is provided a nucleic acid sequence consistingessentially of at least one cas gene, at least one CRISPR spacer and atleast two CRISPR repeats.

In a seventh aspect there is provided a recombinant nucleic acidsequence comprising at least one cas gene and at least two CRISPRrepeats together with at least one CRISPR spacer, wherein the CRISPRspacer is heterologous to the at least one cas gene and/or the at leasttwo CRISPR repeats.

In an eight aspect there is provided a construct comprising one or moreof the nucleic acid sequences described herein

In a ninth aspect there is provided a vector comprising one or more ofthe nucleic acid sequences or one or more of the constructs describedherein.

In an tenth aspect there is provided a cell comprising the nucleic acidsequence or the construct or the vector described herein.

In an eleventh aspect there is provided a method for modulating (e.g.conferring or increasing) the resistance of a cell against a targetnucleic acid or a transcription product thereof comprising the steps of:(i) identifying a sequence (eg. a conserved sequence) in an organism(preferably, a sequence essential to the function or survival of theorganism); (ii) preparing a CRISPR spacer which is homologous to theidentified sequence; (iii) preparing a nucleic acid (eg. a recombinantnucleic acid) comprising at least one cas gene and at least two CRISPRrepeats together with the CRISPR spacer; and (iv) introducing saidnucleic acid into a cell thus to render the cell resistant to saidtarget nucleic acid or transcription product thereof.

In a twelfth aspect there is provided a method for modulating (eg.conferring or increasing) the resistance of a cell against a targetnucleic acid or a transcription product thereof comprising the steps of:(i) identifying one or more CRISPR spacers or pseudo CRISPR spacers inan organism resistant to the target nucleic acid or transcriptionproduct thereof; (ii) preparing a recombinant nucleic acid comprising atleast one cas gene or protein and at least two CRISPR repeats togetherwith said identified one or more spacers; and (iii) introducing saidrecombinant nucleic acid into a cell thus to render the cell resistantto said target nucleic acid or transcription product thereof.

In a thirteenth aspect there is provided a method for modulating (eg.conferring or increasing) the resistance of a cell comprising at leastone or more cas genes or proteins and two or more CRISPR repeats againsta target nucleic acid or a transcription product thereof comprising thesteps of: (i) identifying one or more CRISPR spacers in an organismresistant to the target nucleic acid or transcription product thereof;and (ii) modifying the sequence of one or more CRISPR spacer(s) in thecell such that the CRISPR spacer(s) has homology to the CRISPR spacer(s)in the organism.

In a fourteenth aspect there is provided a method for modulating (eg.reducing or decreasing) the resistance of a cell comprising at least oneor more cas genes or proteins and two or more CRISPR repeats against atarget nucleic acid or a transcription product thereof comprising thesteps of: (i) identifying one or more CRISPR spacers in an organism thatis substantially resistant to the target nucleic acid or transcriptionproduct thereof; and (ii) modifying the sequence of at least one or moreCRISPR spacer(s) in the cell such that the CRISPR spacer(s) has areduced degree of homology to the spacer(s) in the organism.

In a fifteenth aspect there is provided a method for modulating (eg.reducing or decreasing) the resistance of a cell comprising at least oneor more cas genes or proteins and two or more CRISPR repeats against atarget nucleic acid or a transcription product thereof comprisingmodifying the one or more cas genes or proteins and/or two or moreCRISPR repeats in the cell.

In a sixteenth aspect there is provided a method for identifying aCRISPR spacer or pseudo CRISPR spacer for use in modulating theresistance of a cell against a target nucleic acid or a transcriptionproduct thereof comprising the steps of: (i) preparing a cell comprisingat least two CRISPR repeats and at least one cas gene or protein; (ii)identifying at least one CRISPR spacer or pseudo CRISPR spacers in anorganism that is substantially resistant to the target nucleic acid ortranscription product thereof; (iii) modifying the sequence of theCRISPR spacer in the cell such that the CRISPR spacer has homology tothe spacer of the organism; and (iv) determining if the cell modulatesresistance against the target nucleic acid or transcription productthereof, wherein modulation of the resistance of the cell against thetarget nucleic acid or transcription product thereof is indicative thatthe CRISPR spacer modulates the resistance of the cell.

In a seventeenth aspect there is provided a method for identifying a casgene for use in modulating the resistance of a cell against a targetnucleic acid or transcription product thereof comprising the steps of:(i) preparing a cell comprising at least one CRISPR spacer and at leasttwo CRISPR repeats; (ii) engineering the cell such that it comprises atleast one cas gene; and (iii) determining if the cell modulatesresistance against the target nucleic acid or transcription productthereof, wherein modulation of the resistance of the cell against thetarget nucleic acid or transcription product thereof is indicative thatthe cas gene can be used to modulate the resistance of the cell.

In an eighteenth aspect there is provided a method for identifying aCRISPR repeat for use in modulating the resistance of a cell against atarget nucleic acid or transcription product thereof comprising thesteps of: (i) preparing a cell comprising at least one CRISPR spacer andat least one cas gene; (ii) engineering the cell such that it containsthe CRISPR repeat; and (iii) determining if the cell modulatesresistance against the target nucleic acid or transcription productthereof, wherein modulation of the resistance of the cell against thetarget nucleic acid or transcription product thereof is indicative thatthe CRISPR repeat can be used to modulate resistance.

In a nineteenth aspect there is provided a method for identifying afunctional combination of a cas gene and a CRISPR repeat comprising thesteps of: (a) determining the sequences of the cas gene and the CRISPRrepeat; (b) identifying one or more clusters of cas genes as determinedby sequence comparison analysis; (c) identifying one or more clusters ofCRISPR repeats; and (d) combining those cas gene and CRISPR repeatsequences that fall within the same cluster, wherein the combination ofthe cas gene and CRISPR repeat sequences within the same cluster isindicative that the combination is a functional combination.

In a twentieth aspect there is provided a method for modulating thelysotype of a bacterial cell comprising one or more cas genes orproteins and two or more CRISPR repeats comprising the steps of: (i)identifying one or more pseudo CRISPR spacers in the genomic sequence ofa bacteriophage against which resistance is to be modulated; and (ii)modifying the sequence of one or more CRISPR spacers of the bacterialcell such that the CRISPR spacer(s) of the bacterial cell has homologyto the pseudo CRISPR spacer(s) of the bacteriophage against whichresistance is to be modulated.

In a twenty-first aspect there is provided a method for modulating (eg.conferring or increasing) the resistance of a bacterial cell against abacteriophage comprising the steps of: (i) identifying a sequence (eg. aconserved sequence) in a bacteriophage (preferably, a sequence essentialto the function or survival of the bacteriophage); (ii) preparing aCRISPR spacer which is homologous to the identified sequence; (iii)preparing a nucleic acid comprising at least one cas gene and at leasttwo CRISPR repeats together with the CRISPR spacer; and (iv) introducingsaid nucleic acid into the bacterial cell thus to render the bacterialcell resistant to said target nucleic acid or transcription productthereof.

In a twenty-second aspect there is provided a method for modulating (eg.conferring or increasing) the resistance of a bacterial cell against atarget nucleic acid or transcription product in a bacteriophage thereofcomprising the steps of: (i) identifying one or more pseudo CRISPRspacers in a bacteriophage genome that is capable of providingresistance to the target nucleic acid or transcription product thereof;(ii) preparing a recombinant nucleic acid comprising at least one casgene and at least two CRISPR repeats together with said identified oneor more pseudo CRISPR spacers; and (iii) introducing said recombinantnucleic acid into said bacterial cell thus to render the bacterial cellresistant to said target nucleic acid or transcription product thereof.

In a twenty-third aspect there is provided a method for modulating theresistance of a bacterial cell comprising one or more cas genes orproteins and two or more CRISPR repeats against a target nucleic acid ortranscription product thereof in a bacteriophage comprising the stepsof: (i) identifying one or more pseudo CRISPR spacers in a bacteriophagethat is capable of providing resistance to a target nucleic acid ortranscription product thereof; (ii) identifying one or more CRISPRspacers in a bacterial cell in which resistance is to be modulated; and(iii) modifying the sequence of the CRISPR spacer(s) in the bacterialcell in which resistance is to be modulated such that the CRISPRspacer(s) has a higher degree of homology to the pseudo CRISPR spacer(s)of the bacteriophage against which resistance is to be modulated.

In a twenty-fourth aspect there is provided a method for determining theresistance of a cell against a target nucleic acid or a transcriptionproduct thereof comprising identifying one or more functional CRISPRrepeat-cas combinations and one or more CRISPR spacers in the cell.

In a twenty-fifth aspect there is provided a cell obtained or obtainableby the method(s) described herein.

In a twenty-sixth aspect there is provided a CRISPR spacer or pseudoCRISPR spacer obtained or obtainable by the method(s) described herein.

In a twenty-seventh aspect there is provided a cas gene obtained orobtainable by the method(s) described herein.

In a twenty-eighth aspect there is provided a CRISPR repeat obtained orobtainable by the method(s) described herein.

In a twenty-ninth aspect there is provided a functional combinationobtained or obtainable by the method(s) described herein.

In a thirtieth aspect there is provided a recombinant CRISPR locuscomprising a CRISPR spacer or pseudo CRISPR spacer, and/or a cas gene,and/or a CRISPR repeat and/or a functional combination.

In a thirty-first aspect there is provided the use of a cell, a CRISPRspacer or pseudo CRISPR spacer, a cas gene, a CRISPR repeat or afunctional combination for modulating the resistance of a cell against atarget nucleic acid or a transcription product thereof.

In a thirty-second aspect there is provided a cell culture comprising acell, a CRISPR spacer or pseudo CRISPR spacer, a cas gene, a CRISPRrepeat or a functional combination for modulating the resistance of acell against a target nucleic acid or a transcription product thereof.

In a thirty-third aspect there is provided a food product or feedcomprising the culture described herein.

In a thirty-fourth aspect there is provided a process for preparing afood product or feed comprising the use of the culture described herein.

In a thirty-fifth aspect there is provided a food product of feedobtained or obtainable by the process described herein.

In a thirty-sixth aspect there is provided the use of the culturedescribed herein for preparing a food product.

In a thirty-seventh aspect there is provided a nucleotide sequencecomprising or consisting of the sequence set forth in any of SEQ ID Nos.7-10 and SEQ ID Nos. 359-405 or a variant, fragment, homologue orderivative thereof.

In a thirty-eight aspect there is provided an amino acid sequenceencoded by the nucleotide sequence described herein.

In a thirty-ninth aspect there is provided a construct or vectorcomprising one or more of the nucleotide sequences described herein.

In a fortieth aspect there is provided a host cell into which has beenincorporated one or more of the nucleotide sequences described herein orthe construct or vector described herein.

PREFERRED EMBODIMENTS

In some embodiments, the one or more cas genes or proteins are used incombination with two or more CRISPR repeats.

In some embodiments, the one or more cas genes or proteins and/or thetwo or more CRISPR repeats are or are derivable (preferably, derived)from the same cell.

In some embodiments, the one or more cas genes or proteins and the twoor more CRISPR repeats naturally co-occur in the same cell.

In some embodiments, the one or more cas genes or proteins are used incombination with one or more CRISPR spacers.

In some embodiments, the CRISPR spacer(s) is or is derivable(preferably, derived) from an organism that is different to the cellfrom which the one or more cas genes or proteins and/or the two or moreCRISPR repeats are or are derivable (preferably, derived).

In some embodiments, the spacer is obtained from a cell which isresistant to a target nucleic acid.

In some embodiments, the CRISPR spacer is a synthetic nucleic acidsequence.

In some embodiments, the CRISPR spacer(s) have homology to the targetnucleic acid.

In some embodiments, the CRISPR spacer(s) have 100% identity to thetarget nucleic acid over at least the length of the CRISPR spacer core.

In some embodiments, the one or more cas genes or proteins are used incombination with at least one or more CRISPR spacers and at least two ormore CRISPR repeats.

In some embodiments, the target nucleic acid or transcription productthereof is or is derivable (preferably, derived) from bacteriophage DNA.

In some embodiments, the target nucleic acid or transcription productthereof is or is derivable (preferably, derived) from plasmid DNA.

In some embodiments, the target nucleic acid or transcription productthereof is or is derivable (preferably, derived) from a mobile geneticelement.

In some embodiments, the target nucleic acid or transcription productthereof is or is derivable (preferably, derived) from a transposableelement or an insertion sequence.

In some embodiments, the target nucleic acid or transcription productthereof is or is derivable (preferably, derived) from an antibioticresistance gene.

In some embodiments, the target nucleic acid or transcription productthereof is or is derivable (preferably, derived) from a nucleic acidencoding a virulence factor.

In some embodiments, the virulence factor is selected from the groupconsisting of a toxin-, an internalin- and a hemolysin-encoding nucleicacid.

In some embodiments, the one or more cas genes and the two or moreCRISPR repeats are or are derivable (preferably, derived) from the samecell.

In some embodiments, the one or more cas genes and the two or moreCRISPR repeats naturally co-occur in the same cell.

In some embodiments, the CRISPR spacers are or are derivable(preferably, derived) from an organism that is different to the cellfrom which the one or more cas genes and/or the two or more CRISPRrepeats are or are derivable (preferably, derived).

In some embodiments, the cell is a recipient cell or a host cell.

In some embodiments, the one or more cas genes or proteins and/or thetwo or more CRISPR repeats are or are derivable (preferably, derived)from the same cell.

In some embodiments, the spacers are or are derivable (preferably,derived) from an organism that is different to the cell comprising theone or more cas genes or proteins and/or the two or more CRISPR repeats.

In some embodiments, the one or more cas genes or proteins and the twoor more CRISPR repeats naturally co-occur in the same cell.

In some embodiments, said modification comprises inserting one or moreCRISPR spacers and/or pseudo CRISPR spacers into the cell.

In some embodiments, the spacer of the cell has 100% homology to theCRISPR spacer or pseudo CRISPR spacer of the organism.

In some embodiments, said modification comprises genetically engineeringthe CRISPR spacer of the cell.

In some embodiments, all or part of the spacer in the cell is modified.

In some embodiments, said modification comprises the modification of arecombinant spacer.

In some embodiments, said modification occurs through spontaneousmutation or mutagenesis.

In some embodiments, the at least one or more CRISPR spacer(s) in thecell are deleted.

In some embodiments, at least one or more CRISPR repeat(s) in the cellare deleted.

In some embodiments, one or more cas genes are deleted,

In some embodiments, CRISPR and/or one or more cas genes are deleted.

In some embodiments, the one or more cas genes or proteins and/or two ormore CRISPR repeats in the cell are deleted.

In some embodiments, the nucleotide sequences of the cas gene and theCRISPR repeat are or are derivable (preferably, derived) from the sameor different strains.

In some embodiments, the nucleotide sequences of the cas gene and theCRISPR repeat are or are derivable (preferably, derived) from the sameor different species.

In some embodiments, the nucleotide sequences of the cas gene and theCRISPR repeat are or are derivable (preferably, derived) from the sameor different genera.

In some embodiments, the nucleotide sequences of the cas gene and theCRISPR repeat are or are derivable (preferably, derived) from the sameor different organisms.

In some embodiments, the target nucleic acid in the bacteriophage is ahighly conserved nucleic acid sequence.

In some embodiments, the target nucleic acid in the bacteriophageencodes a host specificity protein.

In some embodiments, the target nucleic acid in the bacteriophageencodes a protein that is essential for survival, replication or growthof the bacteriophage.

In some embodiments, the target nucleic acid in the bacteriophageencodes a helicase, a primase, a head or tail structural protein, aprotein with a conserved domain (eg. holin, lysin, and others) or aconserved sequences amongst important phage genes.

In some embodiments, the method for determining the resistance of a cellto a target nucleic acid or a transcription product thereof comprisesthe additional step of comparing the sequence of the one or more CRISPRspacers in the cell with the sequence of the target nucleic acid.

In some embodiments, the method for determining the resistance of a cellto a target nucleic acid or a transcription product thereof comprisesthe additional step of determining the resistance profile of the cell.

In some embodiments, said culture is a starter culture or a probioticculture.

FIGURES

FIG. 1

Schematic representation of CRISPR1 of S. thermophilus CNRZ1066 (42repeats).

FIG. 2

Dotplot analysis of Cas protein sequences (A) and CRISPR locus sequences(B). Organism names (genus, species, strain) are indicated on the rightside of each dotplot (for example Sth_LMG18311=S. thermophilus strainLMG18311).

FIG. 3

Spacer sequences of S. thermophilus CNRZ1066 CRISPR locus were blasted(short nearly exact sequence searches using BlastN at the NCBI website)against the viruses database, and aligned with the subsequent matches inS. thermophilus bacteriophages. (A) The table indicates the spacersequences of CNRZ1066 CRISPR presenting significant sequence identitieswith phage sequences (dark cells). (B) Alignment of the sequence ofinterspacing sequence #29 with eight phage sequences. (Remark: spacer#20 shows similarity to a number of host specificity proteins).

FIG. 4

Putative stem-loop secondary structure of a CRISPR repeat sequence of S.thermophilus. Only one DNA strand is shown.

FIG. 5

Integration of a CRISPR spacer into the CRISPR locus of Streptococcusthermophilus provides resistance against a bacteriophage that the CRISPRspacer shows identity to. The parent DGCC7710 is phage sensitive, andthe BIM DGCC7710RH1 is phage resistant. The BIM DGCC7710RH1 has a newspacer (Sn) in the CRISPR locus, which shows 100% identity to phagesequence. In step (b) the strain is challenged with phage 858 and aphage resistant mutant is selected. In step (c) the CRISPR I locus ofthe mutant has an additional spacer which shares 100% identity withregion 31.921-31.950 bp of the phage.

FIG. 6

Integration of a CRISPR spacer into the CRISPR locus of Streptococcusthermophilus provides resistance against a bacteriophage that the CRISPRspacer shows identity to. The parent DGCC7710 is phage sensitive, andthe BIM DGCC7710RH2 is phage resistant. The BIM DGCC7710 ORH2 has a newspacer (Sn) in the CRISPR locus, which shows 100% identity to phagesequence. In step (b) the strain is challenged with phage 858 and aphage resistant mutant is selected. In step (c) The experiment wasindependently repeated and another mutant was selected. The CRISPR Ilocus of the mutant has an additional spacer (different from that inRH1) which shares 100% identity with region 17.125-17.244 bp of thephage.

FIG. 7

Spacer arrangement of CRISPR I in various Streptococcus thermophilusstrains. Numbers indicate the position of the spacer. Strain names arelisted on the left. Letters indicate CRISPR spacer type, with identicalspacers described with a similar 2-letter code. Spacers with singlenucleotide polymorphisms are labeled with identical letter combination,complemented with a “prime” label. Unique spacers are not described by aletter combination, and are left blank.

FIG. 8

Homology of CRISPR spacers with known sequences, including bacterialchromosomal sequences (shaded in gray), plasmid DNA sequences (blacksquares) and phage genomic sequences (shaded in black).

FIG. 9

A graphical representation of the plasmid system used to geneticallyengineer a number of constructs in Streptococcus thermophilus asdescribed by Russell, M. W., and T. R. Klaenhammer (2001) Efficientsystem for directed integration into the Lactobacillus acidophilus andLactobacillus gasseri chromosomes via homologous recombination. Appliedand Environmental Microbiology 67:4361-4364.

FIG. 10

A graphical representation of the plasmid used to subclone PCR productsof the various constructs described herein (cas1 KO, cas4 KO, RT andS1S2). The plasmid is available commercially from Invitrogen in the TOPOTA Cloning® kit.

FIG. 11

A graphical representation of the plasmid used for homologousrecombination in one embodiment of the present invention.

FIG. 12

A graphical representation illustrating the preparation of the CAS1KOconstruct in which the cas1 gene is disrupted by homologousrecombination.

FIG. 13

A graphical representation illustrating the preparation of the CAS4KOconstruct in which the cas4 gene is disrupted by homologousrecombination.

FIG. 14

A graphical representation illustrating the SIS2 construct engineeringusing specific primers and iterative PCR reactions. The first panelillustrates all primers used and the set up for the first two PCRreactions (reaction #1 with primers P1 and P2 and reaction #2 withprimers P2 and P3). The second panel shows the PCR products obtainedfrom the first two PCR reactions, with the product from reaction #1 onthe left and the product from reaction #2 on the right. The third panelshows the third PCR reaction, using a combination of the products fromthe first two PCRs as the template for the third PCR reaction, andprimer P1 from the first reaction along with primer P4 from the secondreaction. The fourth panel shows the product of PCR#3, which technicallygenerates the S1S2 construct.

FIG. 15

A graphical representation of the details for primer design for primers2 and 3, which contain key sequences for the experiment, derived fromspacers identical to phage sequences (the PCR products derived fromthese PCR primers will generate the spacers that will ultimately provideresistance to the phages).

FIG. 16

A graphical representation of the integration of the S1S2 construct.

FIG. 17

A graphical representation of the preparation of the RT construct usinga restriction enzyme to generate the RT construct from the SIS2construct. There are BglI restriction sites within the repeats allow the“middle” part of the construct to be cut. Following enzymatic digestion,a ligase is used to patch together the two end pieces, thus generating anew construct that has RT, but no spacers.

FIG. 18

A graphical representation of the integration of the RT construct.

FIG. 19

A graphical representation of the RT′ construct.

FIG. 20

A graphical representation of the RT′ construct.

DETAILED DESCRIPTION OF THE INVENTION CRISPR Locus

CRISPR loci are a distinct class of interspersed short sequence repeats(SSRs) that were first recognized in E. coli (Ishino et al. (1987) J.Bacteriol. 169:5429-5433; Nakata et al. (1989) J. Bacteriol.171:3553-3556). Similar interspersed SSRs have been identified inHaloferax mediterranei, Streptococcus pyogenes, Anabaena, andMycobacterium tuberculosis (Groenen et al. (1993) Mol. Microbiol.10:1057-1065; Hoe et al. (1999) Emerg. Infect. Dis. 5:254-263; Masepohlet al. (1996) Biochim. Biophys. Acta 1307:26-30; Mojica et al. (1995)Mol. Microbiol.17:85-93). The CRISPR loci differ from other SSRs by thestructure of the repeats, which have been termed short regularly spacedrepeats (SRSRs) (Janssen et al. (2002) OMICS J. Integ. Biol. 6:23-33;Mojica et al. (2000) Mol. Microbiol. 36:244-246). The repeats are shortelements that occur in clusters, that are always regularly spaced byunique intervening sequences with a constant length (Mojica et al.(2000) Mol. Microbiol. 36:244-246). Although the repeat sequences arehighly conserved between strains, the number of interspersed repeats andthe sequences of the spacer regions differ from strain to strain (vanEmbden et al. (2000) J. Bacteriol. 182:2393-2401).

The common structural characteristics of CRISPR loci are described inJansen. et al. (2002) as (i) the presence of multiple short directrepeats, which show no or very little sequence variation within a givenlocus; (ii) the presence of non-repetitive spacer sequences between therepeats of similar size; (iii) the presence of a common leader sequenceof a few hundred basepairs in most species harbouring multiple CRISPRloci; (iv) the absence of long open reading frames within the locus; and(v) the presence of one or more cas genes.

CRISPRs are typically short partially palindromic sequences of 24-40 bpcontaining inner and terminal inverted repeats of up to 11 bp. Althoughisolated elements have been detected, they are generally arranged inclusters (up to about 20 or more per genome) of repeated units spaced byunique intervening 20-58 bp sequences. CRISPRs are generally homogenouswithin a given genome with most of them being identical. However, thereare examples of heterogeneity in, for example, the Archaea (Mojica etal. 2000).

By way of example, the genome of Streptococcus thermophilus LMG18311contains 3 CRISPR loci; the 36-bp repeated sequences are different inCRISPR1 (34 repeats), CRISPR2 (5 repeats), and CRISPR3 (a singlesequence). Nevertheless, they are perfectly conserved within each locus.CRISPR1 and CRISPR2 repeats are respectively interspaced by 33 and 4sequences of 30 bp in length. All these interspacing sequences aredifferent from each other. They are also different from those found instrain CNRZ1066 (41 interspacing sequences within CRISPR1) and in strainLMD-9 (16 within CRISPR1 and 8 within CRISPR3), which both are S.thermophilus. FIG. 1 describes one of the CRISPRs identified in S.thermophilus.

Various methods for identifying CRISPR loci are already known in theart. By way of example, Jensen et al. (2002) describe a computer basedapproach in which nucleotide sequences are searched for CRISPR motifsusing the PATSCAN program at the server of the Mathematics and ComputerScience Division at the Argonne National Laboratory, Argonne, Ill., USA.The algorithm that was used for identifying CRISPR motifs was p1=a . . .b c . . . d p1 c . . . d p1 c . . . d p1, where a and b are the lowerand upper size limit of the repeat and p1 and c and d are the lower andupper size limit of the spacer sequences. The values of a, b, c and dmay be varied from about 15 to about 70 bp at increments of about 5 bp.

CRISPR loci may be identified using dotplots (using, for example, acomputer program called Dotter).

Sequence similarity analysis may be performed using various methods thatare well known in the art. By way of example, analysis may be performedusing NCBI BLAST with a microbial genomes database(http://www.ncbi.nlm.nih.gov) and GenBank.

The amplification of CRISPR loci has been described in, for example,Mojica et al. (2005) and Pourcel et al. (2005). Amplification of thedesired region of DNA may be achieved by any method known in the art,including polymerase chain reaction (PCR). By “amplification” we meanthe production of additional copies of a nucleic acid sequence. This isgenerally carried out using PCR technologies well known in the art(Dieffenbach and Dveksler (1995) PCR Primer, a Laboratory Manual (ColdSpring Harbor Press, Plainview, N.Y.).

By “polymerase chain reaction” or “PCR” we mean a method such as thatdisclosed in U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202, whichdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. The length of the amplified segment of the desired targetsequence is determined by the relative positions of two oligonucleotideprimers with respect to each other, and therefore, this length is acontrollable parameter. By virtue of the repeating aspect of theprocess, the method is referred to as “PCR”. Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified.”

In the PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify all or part of a CRISPR locus. By “primer” wemean an oligonucleotide, whether occurring naturally as in a purifiedrestriction digest or produced synthetically, which is capable of actingas a point of initiation of synthesis when placed under conditions inwhich synthesis of a primer extension product which is complementary toa nucleic acid strand is induced (i.e., in the presence of nucleotidesand an inducing agent—such as DNA polymerase and at a suitabletemperature and pH). The primer may be single stranded for maximumefficiency in amplification, but may alternatively be double stranded.If double stranded, the primer is first treated to separate its strandsbefore being used to prepare extension products. The primer may be anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer, and the use of the method. PCRprimers are typically at least about 10 nucleotides in length, and mosttypically at least about 20 nucleotides in length.

Methods for designing PCR primers and PCR cloning are generally known inthe art and are disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.). See also Innis et al., eds. (1990) PCR Protocols: AGuide to Methods and Applications (Academic Press, New York); Innis andGelfand, eds. (1995) PCR Strategies (Academic Press, New York); andInnis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, NewYork). Known methods of PCR include, but are not limited to, methodsusing paired primers, nested primers, single specific primers,degenerate primers, gene-specific primers, vector-specific primers,partially mismatched primers, and the like.

The CRISPR loci may comprise, consist or consist essentially of DNA orRNA of genomic, synthetic or recombinant origin.

The CRISPR loci may be double-stranded or single-stranded whetherrepresenting the sense or antisense strand or combinations thereof.

The CRISPR loci may be prepared by use of recombinant DNA techniques(e.g. recombinant DNA), as described herein.

Nucleotide sequences described herein may be obtained fromdatabases—such as GenBank or the JGI website athttp://genome.jgi-psf.org/mic_home.html.

CRISPR Orientation

For the avoidance of doubt, in the context of the present invention theCRISPR locus is orientated as follows.

The CRISPR leader is a conserved DNA segment of defined size. Forexample, the leader sequence of S. thermophilus CRISPR1 is the DNAsegment starting immediately after the stop codon of gene str0660, andending just before the first repeat. The CRISPR leader is located at the5′ end of the CRISPR locus. The CRISPR leader is located immediatelyupstream of the first CRISPR repeat of the CRISPR locus.

The CRISPR trailer is a conserved DNA segment of defined size. Forexample, the trailer sequence of S. thermophilus CRISPR1 is the DNAsegment starting immediately after the terminal repeat, and ending justbefore the stop codon of gene str0661 (located on the opposite DNAstrand). The CRISPR trailer is located at the 3′ end of the CRISPRlocus. The CRISPR trailer is located immediately downstream of theterminal repeat.

By way of example, the CRISPR leader and CRISPR trailer sequences in theCRISPR1 locus of Streptococcus thermophilus strain CNRZ1066 are:

CRISPR leader  5′-CAAGGACAGTTATTGATTTTATAATCACTATGTGGGTATAAAAACGTCAAAATTTCATTTGAG-3′ CRISPR trailer5′-TTGATTCAACATAAAAAGCCAGTTCAATTGAACTTGGCTTT-3′

The CRISPR leader corresponds to positions 625038 to 625100, and theCRISPR trailer corresponds to positions 627845 to 627885 in the fullgenome (CP000024) of Streptococcus thermophilus.

For the avoidance of doubt “upstream” means in the 5′ direction and“downstream” means in the 3′ direction.

CAS

As used herein, the term “cas gene” has the conventional meaning as usedin the art and refers to one or more cas genes that are generallycoupled, associated or close to or in the vicinity of flanking CRISPRloci.

A comprehensive review of the Cas protein family is presented in Haft etal. (2005) Computational Biology 1, 6 e60. As described therein, 41CRISPR-associated (cas) gene families are described, in addition to thefour previously known gene families. It shows that CRISPR systems belongto different classes, with different repeat patterns, sets of genes, andspecies ranges.

The number of cas genes at a given CRISPR locus can vary betweenspecies.

In one aspect, the present invention relates to the use of one or morecas genes or proteins for modulating resistance in a cell (eg. arecipient cell) against a target nucleic acid or a transcription productthereof.

In a further aspect, the present invention relates to the use of one ormore cas genes or proteins and one more CRISPR spacers for modulatingresistance in, a cell (eg. a recipient cell) against a target nucleicacid or a transcription product thereof.

In some embodiments, one or more of the cas genes and/or proteins maynaturally occur in a recipient cell and one or more heterologous spacersmay be integrated or inserted adjacent to the one or more of the casgenes or proteins.

In some embodiments, suitably one or more of the cas genes and/orproteins may be heterologous to the recipient cell and one or more ofthe spacers may be homologous or heterologous. In this instance, thespacers may be integrated or inserted adjacent to the one or more of thecas gene or proteins.

In one aspect, the present invention relates to the use of one or morecas genes or proteins and at least two CRISPR repeats for modulatingresistance in a cell (eg. a recipient cell) against a target nucleicacid or a transcription product thereof.

In one aspect, the present invention relates to the use of one or morecas genes or proteins, at least two CRISPR repeats and at least oneCRISPR spacer for modulating resistance in a cell (eg. a recipient cell)against a target nucleic acid or a transcription product thereof.

CRISPR structures are typically found in the vicinity of four genesnamed cas1 to cas4. The most common arrangement of these genes iscas3-cas4-cas1-cas2. The Cas3 protein appears to be a helicase, whereasCas4 resembles the RecB family of exonucleases and contains acysteine-rich motif, suggestive of DNA binding. Cas1 is generally highlybasic and is the only Cas protein found consistently in all species thatcontain CRISPR loci. Cas2 remains to be characterized. cas1-4 aretypically characterized by their close proximity to the CRISPR loci andtheir broad distribution across bacterial and archaeal species. Althoughnot all cas1-4 genes associate with all CRISPR loci, they are all foundin multiple subtypes.

Bolotin et al. (2005) have recently reported another cluster of threegenes associated with CRISPR structures in many bacterial species, namedhere as cas1B, cas5 and cas6.

The cas gene may be cas1, cas2, cas3, cas4, cas1B, cas5 and/or cas6. Inone embodiment, the cas gene is cas1.

The cas gene may be cas1, cas2, cas3, cas4, cas1B, cas5 and/or cas6 or afragment, variant, homologue or derivative thereof.

The cas genes may be cas1, cas2, cas3, cas4, cas1B, cas5 and/or cas6 ora plurality thereof or a combination thereof—such as cas1 and cas2; cas1and cas3; cas1 and cas4; cas1 and cas1B; cas1 and cas5; cas1 and cas6;cas2 and cas3; cas2 and cas4; cas2 and cas1B; cas2 and cas5; cas2 andcas6; cas3 and cas4; cas3 and cas1B; cas3 and cas5; cas3 and cas6; cas4and cas1B; cas4 and cas5; cas4 and cas6; cas1B and cas5; cas1B and cas6;cas1, cas2 and cas3; cas1, cas2 and cas4; cas1, cas2 and cas1B; cas1,cas2, cas3 and cas4; cas1, cas2, cas3 and cas1B; cas1, cas2, cas3 andcas5; cas1, cas2, cas3 and cas6; cas1, cas2, cas3, cas4 and cas1B; cas1,cas2, cas3, cas4 and cas5; cas1, cas2, cas3, cas4, cas1B and cas6; cas1,cas2, cas3, cas4, cas1B, cas5; cas1, cas2, cas3, cas4, cas1B and cas6;cas1, cas2, cas3, cas4, cas1B, cas5 and cas6; cas2, cas3 and cas4; cas2,cas3 and cas1B; cas2, cas3 and cas5; cas2, cas3 and cas6; cas2, cas3,cas4 and cas1B; cas2, cas3, cas4, and cas5; cas2, cas3, cas4 and cas6;cas2, cas3, cas4, cas1B and cas5; cas2, cas3, cas4, cas1B and cas6;cas2, cas3, cas4, cas1B, cas5 and cas6; cas3, cas4 and cas1B; cas3, cas4and cas5; cas3, cas4 and cas6; cas3, cas4, cas1B and cas5; cas3, cas4,cas1B and cas6; cas3, cas4, cas1B, cas5 and cas6; cas4, cas1B and cas5;cas4, cas1B and cas6; cas4, cas1B, cas5 and cas6; cas5 and cas6 orcombinations thereof.

The cas genes may be cas1 and cas2; cas1 and cas3; cas1 and cas4; cas1and cas1B; cas1 and cas5; cas1 and cas6; cas2 and cas3; cas2 and cas4;cas2 and cas1B; cas2 and cas5; cas2 and cas6; cas3 and cas4; cas3 andcas1B; cas3 and cas5; cas3 and cas6; cas4 and cas1B; cas4 and cas5; cas4and cas6; cas1B and cas5 or cas1B and cas6 or combinations thereof.

The cas genes may be a cas1, cas2 and cas3; cas1, cas2 and cas4; cas1,cas2 and cas1B; cash cas2, cas3 and cas4; cas1, cas2, cas3 and cas1B;cas1, cas2, cas3 and cas5; cas1, cas2, cas3 and cas6; cas1, cas2, cas3,cas4 and cas1B; cas1, cas2, cas3, cas4 and cas5; cas1, cas2, cas3, cas4,cas1B and cas6; cas1, cas2, cas3, cas4, cas1B and cas5; cas1, cas2,cas3, cas4, cas1B and cas6; cas1, cas2, cas3, cas4, cas1B, cas5 and cas6or combinations thereof.

The cas genes may be cas2, cas3 and cas4; cas2, cas3 and cas1B; cas2,cas3 and cas5; cas2, cas3 and cas6; cas2, cas3, cas4 and cas1B; cas2,cas3, cas4, and cas5; cas2, cas3, cas4 and cas6; cas2, cas3, cas4, cas1Band cas5; cas2, cas3, cas4, cas1B and cas6; cas2, cas3, cas4, cas1B,cas5 and cas6 or combinations thereof.

The cas genes may be cas3, cas4 and cas1B; cas3, cas4 and cas5; cas3,cas4 and cas6; cas3, cas4, cas1B and cas5; cas3, cas4, cas1B and cas6;cas3, cas4, cas1B, cas5 and cas6; cas4, cas1B and cas5; cas4, cas1B andcas6; cas4, cas1B, cas5 and cas6; cas5 and cas6 or combinations thereof.

The cas gene may be one or more of cas1, cas2, cas3, cas4, cas1B, cas5and/or cas6 or a plurality thereof—such as a plurality of any 2 casgenes, any 3 cas genes, any 4 cas genes, any 5 cas genes, any 6 casgenes, or any 7 cas genes.

The plurality of cas genes may comprise, consist or consist essentiallyof a plurality of the same cas genes—such as 2 cas genes, 3 cas genes, 4cas genes, 5 cas genes, 6 cas genes, 7 cas genes, 8 cas genes, 9 casgenes, 10 cas genes, 15 cas genes, 20 cas genes, 25 cas genes, 30 casgenes, 35 cas genes, 40 cas genes or even 50 or more cas genes.

The plurality of cas genes may comprise, consist or consist essentiallyof a plurality of different cas genes—such as 2 different cas genes, 3different cas genes, 4 different cas genes, 5 different cas genes, 6different cas genes, 7 different cas genes, 8 different cas genes, 9different cas genes, 10 different cas genes, 15 different cas genes, 20different cas genes, 25 different cas genes, 30 different cas genes, 35different cas genes, 40 different cas genes or even 50 or more differentcas genes.

The plurality of cas genes may comprise, consist or consist essentiallyof a plurality of the same and/or different cas genes—such as 2different cas genes, 3 different cas genes, 4 different cas genes, 5different cas genes, 6 different cas genes, 7 different cas genes, 8different cas genes, 9 different cas genes, 10 different cas genes, 15different cas genes, 20 different cas genes, 25 different cas genes, 30different cas genes, 35 different cas genes, 40 different cas genes oreven 50 or more different cas genes. The same cas gene may be duplicateda plurality of times.

Suitably, the term “cas gene” refers to a plurality of cas genes—such asbetween 2 and 12 cas genes, more preferably, between 3 and 11 cas genes,more preferably, between 4 and 10 cas genes, more preferably, between 4and 9 cas genes, more preferably, between 4 and 8 cas genes, morepreferably, between 4 and 7 cas genes—such as 4, 5, 6, or 7 cas genes.

The cas gene(s) may comprise, consist or consist essentially of DNA orRNA of genomic, synthetic or recombinant origin.

The cas gene(s) may be double-stranded or single-stranded whetherrepresenting the sense or antisense strand or combinations thereof.

The cas gene(s) may be prepared by use of recombinant DNA techniques(e.g. recombinant DNA), as described herein.

As described herein below, the cas gene may be a fragment of a cas gene,thereby indicating hat the cas gene comprises a fraction of a wild-typesequence. Suitably, the sequence comprises at least 30%, at least 40%,at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or least 99% of the wild-type sequence.

For some embodiments it is preferred that the cas gene is the cas genethat is closest to the leader sequence or the first CRISPR repeat at the5′ end of the CRISPR locus—such as cas4 or cas6.

The Cas protein may be Cas, Cas2, Cas3, Cas4, Cas1B, Cas5 and/or Cas6.

The Cas protein may be Cas1, Cas2, Cas3, Cas4, Cas1B, Cas5 and/or Cas6or a fragment, variant, homologue or derivative thereof.

The Cas protein may be Cas1, Cas2, Cas3, Cas4, Cas1B, Cas5 and/or Cas6or a combination thereof—such as Cas1 and Cas2; Cas1 and Cas3; Cas1 andCas4; Cas1 and Cas1B; Cas1 and Cas5; Cas1 and Cas6; Cas2 and Cas3; Cas2and Cas4; Cas2 and Cas1B; Cas2 and Cas5; Cas2 and Cas6; Cas3 and Cas4;Cas3 and Cas1B; Cas3 and Cas5; Cas3 and Cas6; Cas4 and Cas1B; Cas4 andCas5; Cas4 and Cas6; Cas1B and Cas5; Cas1B and Cas6; Cas1, Cas2 andCas3; Cas1, Cas2 and Cas4; Cas1, Cas2 and Cas1B; Cas1, Cas2, Cas3 andCas4; Cas1, Cas2, Cas3 and Cas1B; Cas1, Cas2, Cas3 and Cas5; Cas1, Cas2,Cas3 and Cas6; Cas1, Cas2, Cas3, Cas4 and Cas1B; Cas1, Cas2, Cas3, Cas4and Cas5; Cas1, Cas2, Cas3, Cas4, Cas1B and Cas6; Cas1, Cas2, Cas3,Cas4, Cas1B and Cas5; Cas1, Cas2, Cas3, Cas4, Cas1B and Cas6; Cas1,Cas2, Cas3, Cas4, Cas1B, Cas5 and Cas6; Cas2, Cas3 and Cas4; Cas2, Cas3and Cas1B; Cas2, Cas3 and Cas5; Cas2, Cas3 and Cas6; Cas2, Cas3, Cas4and Cas1B; Cas2, Cas3, Cas4, and Cas5; Cas2, Cas3, Cas4 and Cas6; Cas2,Cas3, Cas4, Cas1B and Cas5; Cas2, Cas3, Cas4, Cas1B and Cas6; Cas2,Cas3, Cas4, Cas1B, Cas5 and Cas6; Cas3, Cas4 and Cas1B; Cas3, Cas4 andCas5; Cas3, Cas4 and Cas6; Cas3, Cas4, Cas1B and Cas5; Cas3, Cas4, Cas1Band Cas6; Cas3, Cas4, Cas1B, Cas5 and Cas6; Cas4, Cas1B and Cas5; Cas4,Cas1B and Cas6 or Cas4, Cas1B, Cas5 and Cas6, Cas5 and Cas6.

The Cas protein may be Cast and Cas2; Cas1 and Cas3; Cast and Cas4; Castand Cas1B; Cas1 and Cas5; Cast and Cas6; Cas2 and Cas3; Cas2 and Cas4;Cas2 and Cas1B; Cas2 and Cas5; Cas2 and Cas6; Cas3 and Cas4; Cas3 andCas1B; Cas3 and Cas5; Cas3 and Cas6; Cas4 and Cas1B; Cas4 and Cas5; Cas4and Cas6; Cas1B and Cas5 or Cas1B and Cas6 or combinations thereof.

The Cas protein may be Cas1, Cas2 and Cas3; Cas1, Cas2 and Cas4; Cas1,Cas2 and Cas1B; Cas1, Cas2, Cas3 and Cas4; Cas1, Cas2, Cas3 and Cas1B;Cas1, Cas2, Cas3 and Cas5; Cas1, Cas2, Cas3 and Cas6; Cas1, Cas2, Cas3,Cas4 and Cas1B; Cas1, Cas2, Cas3, Cas4 and Cas5; Cas1, Cas2, Cas3, Cas4,Cas1B and Cas6; Cast, Cas2, Cas3, Cas4, Cas1B and Cas5; Cas1, Cas2,Cas3, Cas4, Cas1B and Cas6; Cas1, Cas2, Cas3, Cas4, Cas1B, Cas5 and Cas6or combinations thereof.

The Cas protein may be Cas2, Cas3 and Cas4; Cas2, Cas3 and Cas1B; Cas2,Cas3 and Cas5; Cas2, Cas3 and Cas6; Cas2, Cas3, Cas4 and Cas1B; Cas2,Cas3, Cas4, and Cas5; Cas2, Cas3, Cas4 and Cas6; Cas2, Cas3, Cas4, Cas1Band Cas5S; Cas2, Cas3, Cas4, Cas1B and Cas6; Cas2, Cas3, Cas4, Cas1B,Cas5 and Cas6 or combinations thereof.

The Cas protein may be Cas3, Cas4 and Cas1B; Cas3, Cas4 and Cas5; Cas3,Cas4 and Cas6; Cas3, Cas4, Cas1B and Cas5; Cas3, Cas4, Cas1B and Cas6;Cas3, Cas4, Cas1B, Cas5 and Cas6; Cas4, Cas1B and Cas5; Cas4, Cas1B andCas6; Cas4, Cas1B, Cas5 and Cas6; Cas5 and Cas6 or combinations thereof.

The Cas protein may be one or more of Cas1, Cas2, Cas3, Cas4, Cas1B,Cas5 and/or Cas6 or a plurality thereof—such as a plurality of any 2 Casgenes, any 3 Cas genes, any 4 Cas genes, any 5 Cas genes, any 6 Casgenes, or any 7 Cas genes.

The plurality of Cas proteins may comprise, consist or consistessentially of a plurality of the same Cas proteins—such as 2 Casproteins, 3 Cas proteins, 4 Cas proteins, 5 Cas proteins, 6 Casproteins, 7 Cas proteins, 8 Cas proteins, 9 Cas proteins, Cas proteins,15 Cas proteins, 20 Cas proteins, 25 Cas proteins, 30 Cas proteins, 35Cas proteins, 40 Cas proteins or even 50 or more Cas proteins.

The plurality of Cas proteins may comprise, consist or consistessentially of a plurality of different Cas proteins—such as 2 differentCas proteins, 3 different Cas proteins, 4 different Cas proteins, 5different Cas proteins, 6 different Cas proteins, 7 different Casproteins, 8 different Cas proteins, 9 different Cas proteins, 10different Cas proteins, 15 different Cas proteins, 20 different Casproteins, 25 different Cas proteins, 30 different Cas proteins, 35different Cas proteins, 40 different Cas proteins or even 50 or moredifferent Cas proteins.

The plurality of Cas proteins may comprise, consist or consistessentially of a plurality of the same and/or different Casproteins—such as 2 different Cas proteins, 3 different Cas proteins, 4different Cas proteins, 5 different Cas proteins, 6 different Casproteins, 7 different Cas proteins, 8 different Cas proteins, 9different Cas proteins, 10 different Cas proteins, 15 different Casproteins, 20 different Cas proteins, 25 different Cas proteins, 30different Cas proteins, 35 different Cas proteins, 40 different Casproteins or even 50 or more different Cas proteins. The same Casproteins may be duplicated a plurality of times.

Suitably, the term “Cas protein” refers to a plurality of Casproteins—such as between 2 and 12 Cas proteins, more preferably, between3 and 11 Cas proteins, more preferably, between 4 and 10 Cas proteins,more preferably, between 4 and 9 Cas proteins, more preferably, between4 and 8 Cas proteins, more preferably, between 4 and 7 proteinsgenes—such as 4, 5, 6; or 7 Cas proteins.

The Cas protein(s) may be encoded by a cas gene which may comprise DNAor RNA of genomic, synthetic or recombinant origin.

The Cas protein(s) may be encoded by a cas gene which may bedouble-stranded or single-stranded whether representing the sense orantisense strand or combinations thereof.

The Cas protein(s) may be prepared by use of recombinant DNA techniques(e.g. recombinant DNA), as described herein.

In a further aspect, there is provided a method for identifying a casgene for use in modulating the resistance of a cell against a targetnucleic acid or transcription product thereof comprising the steps of:(i) preparing a cell comprising at least one CRISPR spacer and at leasttwo CRISPR repeats; (ii) engineering the cell such that it comprises atleast one cas gene; and (iii) determining if the cell modulatesresistance against the target nucleic acid or transcription productthereof, wherein modulation of the resistance of the cell against thetarget nucleic acid or transcription product thereof is indicative thatthe cas gene can be used to modulate the resistance of the cell.

One or more of the cas genes may be used to engineer a cell—such as arecipient cell. In particular, one or more cas genes may be used toengineer a cell—such as a recipient cell—that in combination with one ormore, preferably, two or more CRISPR repeats and one or more CRISPRspacers can be used to modulate the resistance of a cell against atarget nucleic acid or a transcription product thereof. By way ofexample, the cas gene(s) may be inserted into the DNA of a cell (eg. arecipient cell)—such as plasmid DNA or genomic DNA of a cell—usingvarious methods that are well known in the art. By way of furtherexample, the cas genes may be used as a template upon which to modify(eg. mutate) the DNA of a cell (eg. a recipient cell)—such as plasmidDNA or genomic DNA—such that cas genes are created or formed in the DNAof the cell. By way of further example, the cas genes may be cloned intoa construct, a plasmid or a vector and the like which is thentransformed into the cell, using methods such as those described herein.

The cas genes may comprise or consist of a cas cluster selected from thegroup consisting of any one or more of SEQ ID No. 461, SEQ ID No. 466,SEQ ID No. 473, SEQ ID No. 478, SEQ ID No. 488, SEQ ID No. 493, SEQ IDNo. 498, SEQ ID No. 504, SEQ ID No. 509, SEQ ID No. 517

The cas genes may comprise or consist of any one or more of SEQ ID Nos.462-465, 467-472, 474-477, 479-487, 489-492, 494-497, 499-503, 505-508,510-516 and/or 517-521.

Suitably, the one or more cas genes or proteins are used together withor in combination with one or more, preferably, two or more CRISPRrepeats and optionally one or more CRISPR spacers.

CRISPR Repeat

As used herein, the term “CRISPR repeat” has the conventional meaning asused in the art ie. multiple short direct repeats, which show no or verylittle sequence variation within a given CRISPR locus.

As used herein, the term “CRISPR” is synonymous with the term “CRISPRrepeat”.

The number of nucleotides in a repeat is generally about 20 to about 40base pairs, but may be about 20 to about 39 base pairs, about 20 toabout 37 base pairs, about 20 to about 35 base pairs, about 20 to about33 base pairs, about 20 to about 30 base pairs, about 21 to about 40base pairs, about 21 to about 39 base pairs, about 21 to about 37 basepairs, about 23 to about 40 base pairs, about 23 to about 39 base pairs,about 23 to about 37 base pairs, about 25 to about 40 base pairs, about25 to about 39 base pairs, about 25 to about 37 base pairs, about 25 toabout 35 base pairs, or about 28 or 29 base pairs. The number of repeatsmay range from about 1 to about 140, from about 1 to about 100, fromabout 2 to about 100, from about 5 to about 100, from about 10 to about100, from about 15 to about 100, from about 20 to about 100, from about25 to about 100, from about 30 to about 100, from about 35 to about 100,from about 40 to about 100, from about 45 to about 100, from about 50 toabout 100, from about 1 to about 135, from about 1 to about 130, fromabout 1 to about 125, from about 1 to about 120, from about 1 to about115, from about 1 to about 110, from about 1 to about 105, from about 1to about 100, from about 1 to about 95, from about 1 to about 90, fromabout 1 to about 80, from about 1 to about 70, from about 1 to about 60,from about 1 to about 50, from about 10 to about 140, from about 10 toabout 130, from about 10 to about 120, from about 10 to about 110, fromabout 10 to about 95, from about 10 to about 90, from about 20 to about80, from about 30 to about 70, from about 30 to about 60, from about 30to about 50, from about 30 to about 40, or about 32.

Suitably, the number of nucleotides in a repeat is generally about 20 toabout 40 base pairs, but may be about 20 to about 39 base pairs, about20 to about 37 base pairs, about 20 to about 35 base pairs, about 20 toabout 33 base pairs, about 20 to about 30 base pairs, about 21 to about40 base pairs, about 21 to about 39 base pairs, about 21 to about 37base pairs, about 23 to about 40 base pairs, about 23 to about 39 basepairs, about 23 to about 37 base pairs, about 25 to about 40 base pairs,about 25 to about 39 base pairs, about 25 to about 37 base pairs, about25 to about 35 base pairs, or about 28 or 29 base pairs.

Suitably, the number of repeats may range from about 2 to about 140,from about 2 to about 100, from about 2 to about 100, from about 5 toabout 100, from about 10 to about 100, from about 15 to about 100, fromabout 20 to about 100, from about 25 to about 100, from about 30 toabout 100, from about 35 to about 100, from about 40 to about 100, fromabout 45 to about 100, from about 50 to about 100.

Suitably, the number of repeats may range from about 2 to about 135,from about 2 to about 130, from about 2 to about 125, from about 2 toabout 120, from about 2 to about 115, from about 2 to about 110, fromabout 2 to about 105, from about 2 to about 100, from about 2 to about95, from about 2 to about 90, from about 2 to about 80, from about 2 toabout 70, from about 2 to about 60, from about 2 to about 50, from about2 to about 40, from about 2 to about 30, from about 2 to about 20, fromabout 2 to about 10, from about 2 to about 9, from about 2 to about 8,from about 2 to about 7, from about 2 to about 6, from about 2 to about5, from about 2 to about 4, or from about 2 to about 3.

The CRISPR repeat(s) may comprise, consist or consist essentially of DNAor RNA of genomic, synthetic or recombinant origin.

The CRISPR repeat(s) may be double-stranded or single-stranded whetherrepresenting the sense or antisense strand or combinations thereof.

The CRISPR repeat(s) may be prepared by use of recombinant DNAtechniques (e.g. recombinant DNA), as described herein.

One or more of the CRISPR repeats may be used to engineer a cell—such asa recipient cell. In particular, one or more, preferably, two or moreCRISPR repeats may be used to engineer a cell—such as a recipientcell—that in combination with one or more cas genes or proteins and oneor more CRISPR spacers can be used to modulate the resistance of a cellagainst a target nucleic acid or a transcription product thereof. By wayof example, the CRISPR repeat(s) may be inserted into the DNA of a cell(eg. a recipient cell)—such as plasmid DNA or genomic DNA of acell—using various methods that are well known in the art. By way offurther example, the CRISPR repeat(s) may be used as a template uponwhich to modify (eg. mutate) the DNA of a cell (eg. a recipientcell)—such as plasmid DNA or genomic DNA—such that CRISPR repeat(s) arecreated or engineered in the DNA of the cell. By way of further example,CRISPR repeat(s) may be cloned into a construct, a plasmid or a vectorand the like which is then transformed into the cell, using methods suchas those described herein.

In a further aspect of the present invention, there is also provided amethod for identifying a CRISPR repeat for use in modulating theresistance of a cell against a target nucleic acid or transcriptionproduct thereof comprising the steps of: (i) preparing a cell comprisingat least one CRISPR spacer and at least one cas gene; (ii) engineeringthe cell such that it contains a CRISPR repeat; and (iii) determining ifthe cell modulates resistance against the target nucleic acid ortranscription product thereof, wherein modulation of the resistance ofthe cell against the target nucleic acid or transcription productthereof is indicative that the CRISPR repeat can be used to modulateresistance.

Suitably, one or more cas genes or proteins are used together with or incombination with one or more, preferably, two or more CRISPR repeats andoptionally one or more CRISPR spacers. Suitably, the cas gene(s) orprotein(s) and CRISPR repeat(s) form a functional combination asdescribed below.

A CRISPR spacer is flanked by two CRISPR repeats. In other words, aCRISPR spacer has at least one CRISPR repeat on each side.

The CRISPR repeats may comprise or consist of the nucleotide sequenceset forth in any one or more of SEQ ID Nos. 1-22.

Functional Combination

As mentioned above, surprisingly, the inventors have discovered that agiven set of cas genes or proteins is always associated with a givenrepeated sequence within a particular CRISPR locus. In other words, casgenes or proteins seem to be specific for a given DNA repeat, meaningthat cas genes or proteins and the repeated sequence form a functionalpair.

Accordingly, particular combinations of one or more cas genes orproteins and one or more, preferably, two or more CRISPR repeats areused in order for a CRISPR spacer to confer resistance against a targetnucleic acid or transcription product thereof in a cell (eg. a recipientcell). Accordingly, it has been surprisingly found that it is notpossible to merely use any cas genes or proteins or any CRISPR repeat.Instead it is a feature of the present invention that the combination isfunctional.

In the context of the CRISPR repeat-cas gene or protein combinationdescribed herein, the term “functional” means that the combination isable to confer resistance to a target nucleic acid or a transcriptionproduct thereof when used together with a CRISPR spacer which alignswith or is homologous to a target nucleic acid or transcription productthereof.

As used herein the term “functional CRISPR repeat-cas combination” and“functional CRISPR repeat-cas gene combination” includes a functionalcombination in which cas is a cas gene or a Cas protein.

Suitably, the one or more cas genes or proteins and/or the one or more,preferably, two or more CRISPR repeats are or are derivable (preferably,derived) from the same cell (eg. the same recipient cell).

In one embodiment, the term “derivable” is synonymous with the term“obtainable”.

In one embodiment, the term “derived” is synonymous with the term“obtained”.

Suitably, the one or more cas genes or proteins and/or the one or more,preferably, two or more CRISPR repeats are derivable (preferably,derived) from the same CRISPR locus within a genome or plasmid,preferably a genome or plasmid of the same strain, species or genera.

Suitably, the one or more cas genes or proteins and/or the one or more,preferably, two or more CRISPR repeats are derivable (preferably,derived) from the same CRISPR locus within a single genome or plasmid,preferably a single genome or plasmid of the same strain, species orgenera.

Suitably, the one or more cas genes or proteins and the one or more,preferably, two or more CRISPR repeats naturally co-occur.

Suitably, the one or more cas genes or proteins and the one or more,preferably, two or more CRISPR repeats naturally co-occur in the samecell (eg. recipient cell).

Suitably, the one or more cas genes or proteins and the one or more,preferably, two or more CRISPR repeats naturally co-occur in the samegenome of a cell (eg. recipient cell).

Suitably, the one or more cas genes or proteins and the one or more,preferably, two or more CRISPR repeats naturally co-occur in the samegenome of a strain, species or genera.

Accordingly, in a further aspect, there is provided a combination ornucleic acid consisting essentially of at least two CRISPR repeats andat least one cas gene or protein.

In one embodiment, the term “consists essentially of” refers to acombination of at least two CRISPR repeats and at least one cas gene orprotein and excluding at least one further component of a CRISPRlocus—such as the absence of one or more CRISPR spacer(s) and/or theabsence of one or more common leader sequence(s) of a CRISPR locus.

In one embodiment, the term “consists essentially of” refers to acombination of at least two CRISPR repeats and at least one cas gene orprotein only and excluding all other components of a CRISPR locus—suchas a naturally occurring CRISPR locus.

In a further embodiment, the term “consists essentially of” refers to acombination of at least two CRISPR repeats and at least one cas gene orprotein only and excluding at least one further component of a CRISPRlocus—preferably excluding at least one further component of a naturallyoccurring CRISPR locus.

In a further embodiment, the term “consists essentially of” refers to acombination of at least two CRISPR repeats and at least one cas gene orprotein with the proviso that at least one further component of thenatural CRISPR locus is absent (eg. substantially absent).

Suitably, there is provided a combination of at least two CRISPR repeatsand at least one cas gene or protein with the proviso that all othercomponents of the CRISPR locus are absent (eg. substantially absent),preferably that all other components of the CRISPR locus of the naturalcombination of CRISPR repeat(s) and cas gene(s) are absent.

Suitably, the one or more cas genes or proteins are used in combinationor together with one or more CRISPR spacers.

Suitably, the one or more cas genes or proteins are used in combinationor together with at least one or more CRISPR spacers and at least one ormore, preferably, two or more CRISPR repeats.

In one embodiment, the CRISPR spacer(s) are or are derivable(preferably, derived) from an organism (eg. a donor organism) that isdifferent to the cell (eg. the recipient cell) from which the one ormore cas genes or proteins and/or the one or more, preferably, two ormore CRISPR repeats are or are derivable (preferably, derived).

Various arrangements of CRISPR repeats(s) and cas gene(s) orprotein(s)—such as functional CRISPR repeat-cas combinations—arecontemplated.

The combination may comprise, consist or consist essentially of at leastany of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 CRISPR repeat (s)in combination with any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 cas genes or proteins—such as 16 CRISPRrepeat and 12 cas genes or proteins or 18 CRISPR repeats and 20 casgenes or proteins or any other combinations thereof.

The CRISPR repeat(s) and cas gene(s) may be arranged in various ways.

The combination may be cas1-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats),cas2-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats), cas3-repeat (whereinthe repeat is at least two repeats, preferably, with at least one spacerin between the repeats), cas4-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats),cas1B-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats), cas5-repeat (whereinthe repeat is at least two repeats, preferably, with at least one spacerin between the repeats), and/or cas6-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats).

The cas gene may be cas1, cas2, cas3, cas4, cas1B, cas5 and/or cas6 or afragment, variant, homologue or derivative thereof.

The cas genes may be cas1, cas2, cas3, cas4, cas1B, cas5 and/or cas6 ora plurality thereof or a combination thereof—such as cas1 andcas2-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats); cas1 and cas3-repeat(wherein the repeat is at least two repeats, preferably, with at leastone spacer in between the repeats); cas1 and cas4-repeat (wherein therepeat is at least two repeats, preferably, with at least one spacer inbetween the repeats); cas1 and cas1B-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas) and cas5-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas1 and cas6-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas2 andcas3-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats); cas2 and cas4-repeat(wherein the repeat is at least two repeats, preferably, with at leastone spacer in between the repeats); cas2 and cas1B-repeat (wherein therepeat is at least two repeats, preferably, with at least one spacer inbetween the repeats); cas2 and cas5-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas2 and cas6-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas3 and cas4-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas3 andcas1B-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats); cas3 and cas5-repeat(wherein the repeat is at least two repeats, preferably, with at leastone spacer in between the repeats); cas3 and cas6-repeat (wherein therepeat is at least two repeats, preferably, with at least one spacer inbetween the repeats); cas4 and cas1B-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas4 and cas5-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas4 and cas6-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas1B andcas5-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats); cas1B and cas6-repeat(wherein the repeat is at least two repeats, preferably, with at leastone spacer in between the repeats); cas1, cas2 and cas3-repeat (whereinthe repeat is at least two repeats, preferably, with at least one spacerin between the repeats); cas1, cas2 and cas4-repeat (wherein the repeatis at least two repeats, preferably, with at least one spacer in betweenthe repeats); cas1, cas2 and cas1B-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas1, cas2, cas3 and cas4-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas1 cas2, cas3 and cas1B-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas1, cas2, cas3 and cas5-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas1, cas2, cas3 and cas6-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas1, cas2, cas3, cas4 and cas1B-repeat (wherein the repeat isat least two repeats, preferably, with at least one spacer in betweenthe repeats); cas1, cas2, cas3, cas4 and cas5-repeat (wherein the repeatis at least two repeats, preferably, with at least one spacer in betweenthe repeats); cas1, cas2, cas3, cas4, cas1B and cas6-repeat (wherein therepeat is at least two repeats, preferably, with at least one spacer inbetween the repeats); cas1, cas2, cas3, cas4, cas1B, cas5-repeat(wherein the repeat is at least two repeats, preferably, with at leastone spacer in between the repeats); cas1, cas2, cas3, cas4, cas1B andcas6-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats); cas1, cas2, cas3,cas4, cas1B, cas5 and cas6-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas2, cas3 and cas4-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas2, cas3and cas1B-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas2, cas3and cas5-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats): cas2, cas3 andcas6-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats); cas2, cas3, cas4 andcas1B-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats); cas2, cas3, cas4, andcas5-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats); cas2, cas3, cas4 andcas6-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats); cas2, cas3, cas4,cas1B and cas5-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas2,cas3, cas4, cas1B and cas6-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas2, cas3, cas4, cas1B, cas5 and cas6-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas3, cas4 and cas1B-repeat (wherein the repeat is at leasttwo repeats, preferably, with at least one spacer in between therepeats); cas3, cas4 and cas5-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas3, cas4 and cas6 repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas3,cas4, cas1B and cas5-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas3,cas4, cas1B and cas6-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas3,cas4, cas1B, cas5 and cas6-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas4, cas1B and cas5-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas4,cas1B and cas6-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas4,cas1B, cas5 and cas6-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas5 andcas6-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats) or combinationsthereof.

The cas genes may be cas1 and cas2-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas1 and cas3-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas1 and cas4-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas1 andcas1B-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats); cas1 and cas5-repeat(wherein the repeat is at least two repeats, preferably, with at leastone spacer in between the repeats); cas1 and cas6-repeat (wherein therepeat is at least two repeats, preferably, with at least one spacer inbetween the repeats); cas2 and cas3-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas2 and cas4-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas2 and cas1B-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas2 andcas5-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats); cas2 and cas6-repeat(wherein the repeat is at least two repeats, preferably, with at leastone spacer in between the repeats); cas3 and cas4-repeat (wherein therepeat is at least two repeats, preferably, with at least one spacer inbetween the repeats); cas3 and cas1B-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas3 and cas5-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas3 and cas6-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas4 andcas1B-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats); cas4 and cas5-repeat(wherein the repeat is at least two repeats, preferably, with at leastone spacer in between the repeats); cas4 and cas6-repeat (wherein therepeat is at least two repeats, preferably, with at least one spacer inbetween the repeats); cas1B and cas5-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats) or cas1B and cas6-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats) orcombinations thereof.

The cas genes may be a cas1, cas2 and cas3-repeat (wherein the repeat isat least two repeats, preferably, with at least one spacer in betweenthe repeats); cas1, cas2 and cas4-repeat (wherein the repeat is at leasttwo repeats, preferably, with at least one spacer in between therepeats); cas1, cas2 and cas1B-repeat (wherein the repeat is at leasttwo repeats, preferably, with at least one spacer in between therepeats); cas1, cas2, cas3 and cas4-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas1, cas2, cas3 and cas1B-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas1, cas2, cas3 and cas5-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas1, cas2, cas3 and cas6-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas1, cas2, cas3, cas4 and cas1B-repeat (wherein the repeat isat least two repeats, preferably, with at least one spacer in betweenthe repeats); cas1, cas2, cas3, cas4 and cas5-repeat (wherein the repeatis at least two repeats, preferably, with at least one spacer in betweenthe repeats); cas1, cas2, cas3, cas4, cas1B and cas6-repeat (wherein therepeat is at least two repeats, preferably, with at least one spacer inbetween the repeats); cas1, cas2, cas3, cas4, cas1B and cas5-repeat(wherein the repeat is at least two repeats, preferably, with at leastone spacer in between the repeats); cas1, cas2, cas3, cas4, cas1B andcas6-repeat (wherein the repeat is at least two repeats, preferably,with at least one spacer in between the repeats); cas1, cas2, cas3,cas4, cas1B, cas5 and cas6-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats) orcombinations thereof.

The cas genes may be cas2, cas3 and cas4-repeat (wherein the repeat isat least two repeats, preferably, with at least one spacer in betweenthe repeats); cas2, cas3 and cas1B-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas2, cas3 and cas5-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas2, cas3 and cas6-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas2,cas3, cas4 and cas1B-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas2,cas3, cas4, and cas5-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas2,cas3, cas4 and cas6-repeat (wherein the repeat is at least two repeats,preferably, with at least one spacer in between the repeats); cas2,cas3, cas4, cas1B and cas5-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas2, cas3, cas4, cas1B and cas6-repeat (wherein the repeat is at leasttwo repeats, preferably, with at least one spacer in between therepeats); cas2, cas3, cas4, cas1B, cas5 and cas6-repeat (wherein therepeat is at least two repeats, preferably, with at least one spacer inbetween the repeats) or combinations thereof.

The cas genes may be cas3, cas4 and cas1B-repeat (wherein the repeat isat least two repeats, preferably, with at least one spacer in betweenthe repeats); cas3, cas4 and cas5-repeat (wherein the repeat is at leasttwo repeats, preferably, with at least one spacer in between therepeats); cas3, cas4 and cas6-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas3, cas4, cas1B and cas5-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas3, cas4, cas1B and cas6-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats);cas3, cas4, cas1B, cas5 and cas6-repeat (wherein the repeat is at leasttwo repeats, preferably, with at least one spacer in between therepeats); cas4, cas1B and cas5-repeat (wherein the repeat is at leasttwo repeats, preferably, with at least one spacer in between therepeats); cas4, cas1B and cas6-repeat (wherein the repeat is at leasttwo repeats, preferably, with at least one spacer in between therepeats); cas4, cas1B, cas5 and cas6-repeat (wherein the repeat is atleast two repeats, preferably, with at least one spacer in between therepeats); cas5 and cas6-repeat (wherein the repeat is at least tworepeats, preferably, with at least one spacer in between the repeats) orcombinations thereof.

Where the combination of a cas gene and a CRISPR repeat comprises morethan one cas gene, it will be understood that the CRISPR repeat may beinserted at the 3′ end of the cas genes, the 5′ end of the cas genes, oreven in between the cas genes, provided that at least one of the casgenes remains functional.

In one embodiment, a first CRISPR repeat-cas gene or protein combination(comprising at least one cas gene or protein and at least two CRISPRrepeats, wherein both are derivable (preferably, derived) from the sameCRISPR locus within a genome) may be used in combination with a secondCRISPR repeat-cas gene or protein combination (comprising at least onecas gene or protein and at least two CRISPR repeats, wherein both arederivable (preferably, derived) from the same or a different CRISPRlocus within a genome). Accordingly, in this embodiment of theinvention, the first and second combination are derivable (preferably,derived) from the same or different CRISPR loci within a genome.

Thus the first and second CRISPR repeat-cas gene or protein combinationsmay even be from different genomes—such as different genomes within thesame cluster, as described in further detail herein.

In a still further embodiment of the present invention, a first and/or asecond CRISPR repeat-cas gene or protein combination (comprising atleast one cas gene and at least two CRISPR repeats derivable(preferably, derived) from the same CRISPR locus within a genome) may beused in combination with 3, 4, 5, 6, 7, 8, 9 or 10 or more CRISPRrepeat-cas gene or protein combinations (each comprising at least onecas gene or protein and at least two CRISPR repeats derivable(preferably, derived) from the same or a different CRISPR loci within agenome). Accordingly, in this embodiment of the invention, thecombinations are derivable (preferably, derived) from the same ordifferent CRISPR loci within a genome.

In a further embodiment of the invention, the combinations may even befrom different genomes—such as different genomes within the samecluster, as described in further detail herein.

In other words, for the CRISPR-repeat-cas gene or protein combination toconfer resistance, in some embodiments, the CRISPR-repeat(s) and casgene(s) or protein(s) naturally co-occur within a given CRISPR locus ofa genome. In some embodiments, the CRISPR-repeat(s) and cas gene(s) orprotein(s) naturally co-occur within the same CRISPR locus of a genome.These functional combinations together may confer resistance against atarget nucleic acid or a transcription product thereof.

In a further aspect, there is provided a method for identifying afunctional combination of a cas gene or protein and a CRISPR repeatcomprising the steps of: (i) analysing the sequences (eg. nucleic acidor protein sequences) of the cas gene or protein and the CRISPR repeat;(ii) identifying one or more clusters of cas genes or proteins; (iii)identifying one or more clusters of CRISPR repeats; and (iv) combiningthose cas gene or protein and CRISPR repeat sequences that fall withinthe same cluster.

In a further aspect, there is provided a method for identifying afunctional combination of a cas gene or protein and a CRISPR repeat foruse in modulating the resistance of a cell against a target nucleic acidor a transcription product thereof comprising the steps of: (i)preparing a cell comprising a combination of one or more cas genes orproteins and one or more, preferably, two or more CRISPR repeats; (ii)engineering the cell such that it contains one or more CRISPR spacers;and (iii) determining if the cell modulates resistance against a targetnucleic acid, wherein modulation of the resistance of The cell againstthe target nucleic acid or a transcription product thereof is indicativethat the combination can be used to modulate the resistance of the cellagainst the target nucleic acid.

Suitably, the sequences of the cas gene or protein and the CRISPR repeatare or are derivable (preferably, derived) from the same or differentstrains.

Suitably, the sequences of the cas gene or protein and the CRISPR repeatare or are derivable (preferably, derived) from the same or differentspecies.

Suitably, the sequences of the cas gene or protein and the CRISPR repeatare or are derivable (preferably, derived) from the same or differentgenera.

Suitably, the sequences of the cas gene or protein and the CRISPR repeatare or are derivable (preferably, derived) from the same or differentorganisms.

Suitably, the analysis is performed using dotplot analysis.

The combination may comprise, consist or consist essentially of DNA orRNA of genomic, synthetic or recombinant origin.

The combination may comprise, consist or consist essentially of DNA andRNA of genomic, synthetic or recombinant origin.

The combination may comprise, consist or consist essentially of a DNACRISPR repeat of genomic, synthetic or recombinant origin.

The combination may comprise, consist or consist essentially of a RNACRISPR repeat of genomic, synthetic or recombinant origin.

The combination may comprise, consist or consist essentially of a DNAcas gene repeat of genomic, synthetic or recombinant origin.

The combination may comprise, consist or consist essentially of a RNAcas gene of genomic, synthetic or recombinant origin.

The combination may comprise, consist or consist essentially of a DNACRISPR repeat and DNA cas gene of genomic, synthetic or recombinantorigin.

The combination may comprise, consist or consist essentially of a DNACRISPR repeat and RNA cas gene of genomic, synthetic or recombinantorigin.

The combination may comprise, consist or consist essentially of a RNACRISPR repeat and DNA cas gene of genomic, synthetic or recombinantorigin.

The combination may comprise, consist or consist essentially of a RNACRISPR repeat and RNA cas gene of genomic, synthetic or recombinantorigin.

The CRISPR repeat may be double-stranded or single-stranded whetherrepresenting the sense or antisense strand or combinations thereof.

The cas gene may be double-stranded or single-stranded whetherrepresenting the sense or antisense strand or combinations thereof.

The CRISPR repeat may be double-stranded or single-stranded whetherrepresenting the sense or antisense strand or combinations thereof andthe cas gene may be double-stranded or single-stranded whetherrepresenting the sense or antisense strand or combinations thereof.

The CRISPR repeat may be double-stranded whether representing the senseor antisense strand or combinations thereof and the cas gene may bedouble-stranded whether representing the sense or antisense strand orcombinations thereof.

The CRISPR repeat may be double-stranded whether representing the senseor antisense strand or combinations thereof and the cas gene may besingle-stranded whether representing the sense or antisense strand orcombinations thereof.

The CRISPR repeat may be single-stranded whether representing the senseor antisense strand or combinations thereof and the cas gene may bedouble-stranded whether representing the sense or antisense strand orcombinations thereof.

The CRISPR repeat may be single-stranded whether representing the senseor antisense strand or combinations thereof and the cas gene may besingle-stranded whether representing the sense or antisense strand orcombinations thereof.

One or more of the functional combinations as described above may beused to engineer a cell—such as a recipient cell. In particular, one ormore functional combinations may be used to engineer a cell—such as arecipient cell—that in combination with one or more CRISPR spacers canbe used to modulate the resistance of a cell against a target nucleicacid or a transcription product thereof. By way of example, thefunctional combinations may be inserted into the DNA of a cell (eg. arecipient cell)—such as plasmid DNA or genomic DNA of a cell—usingvarious methods that are well known in the art. By way of furtherexample, the functional combinations may be used as a template uponwhich to modify (eg. mutate) the DNA of a cell (eg. a recipientcell)—such as plasmid DNA or genomic DNA—such that functionalcombinations are created in the DNA of the cell. By way of furtherexample, functional combinations may be cloned into a construct, aplasmid or a vector and the like which are then transformed into thecell, using methods such as those described herein.

In one embodiment, the functional combination is obtained or obtainableby a method comprising the steps of: (a) analysing the sequences of acas gene and a CRISPR repeat; (b) identifying one or more clusters ofcas genes; (c) identifying one or more clusters of CRISPR repeats; and(d) combining those cas gene and CRISPR repeat sequences that fallwithin the same cluster, wherein the combination of the cas gene andCRISPR repeat sequences within the same cluster is indicative that thecombination is a functional combination.

Clusters are described in further detail below.

CRISPR Spacer

As used herein, the term “CRISPR spacer” has the conventional meaning asused in the art and refers to the non-repetitive spacer sequences thatare found between multiple short direct repeats (i.e. CRISPR repeats) ofCRISPR loci. In other words, a CRISPR spacer is found between two CRISPRrepeats.

It has been found that CRISPR spacer sequences in prokaryotes often havesignificant similarities to a variety of DNA molecules—such as geneticelements (including, but not limited to, chromosomes, bacteriophages,and conjugative plasmids). Interestingly, cells carrying these CRISPRspacers are unable to be infected by DNA molecules containing sequenceshomologous to the spacers (Mojica et al. 2005).

Typically, the CRISPR spacer is naturally present in between twoidentical multiple short direct repeats that are palindromic.

Suitably, the CRISPR spacer is homologous to the target nucleic acid ora transcription product thereof or an identified sequence. Althoughhomology can also be considered in terms of similarity, in the contextof the present invention it is preferred to express homology in terms ofsequence identity. A homologous sequence is taken to include a CRISPRspacer, which may be at least 70, 75, 80, 85 or 90% identical, or atleast 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the targetnucleic acid sequence or a transcription product thereof or anidentified sequence.

In some embodiments, the CRISPR spacer is 100% identical to the targetnucleic acid sequence.

The number of CRISPR spacers at a given CRISPR loci or locus can varybetween species.

Suitably, the number of spacers may range from about 1 to about 140,from about 1 to about 100, from about 2 to about 100, from about 5 toabout 100, from about 10 to about 100, from about 15 to about 100, fromabout 20 to about 100, from about 25 to about 100, from about 30 toabout 100, from about 35 to about 100, from about 40 to about 100, fromabout 45 to about 100, from about 50 to about 100.

Suitably, the number of spacers may range from about 1 to about 135,from about 1 to about 130, from about 1 to about 125, from about 1 toabout 120, from about 1 to about 115, from about 1 to about 110, fromabout 1 to about 105, from about 1 to about 100, from about 1 to about95, from about 1 to about 90, from about 1 to about 80, from about 1 toabout 70, from about 1 to about 60, from about 1 to about 50, from about1 to about 40, from about 1 to about 30, from about 1 to about 20, fromabout 1 to about 10, from about 1 to about 9, from about 1 to about 8,from about 1 to about 7, from about 1 to about 6, from about 1 to about5, from about 1 to about 4, from about 1 to about 3, from about 1 toabout 2.

Typically, CRISPR spacers are identified by sequence analysis as the DNAstretches located in between two repeats.

As described herein, the inventors have surprisingly discovered that theuse of one or more cas genes or proteins in combination with one ormore, preferably, two or more CRISPR repeats (preferably, functionalcombination(s) thereof) provides for the specificity of immunity to beconferred by one or more CRISPR spacers in a cell—such as a recipientcell.

As used herein, the term “specificity of immunity” means that immunitycan be conferred against a specific nucleic acid sequence ortranscription product thereof using a specific CRISPR spacer (or pseudoCRISPR spacer) sequence. Accordingly, a given CRISPR spacer does notconfer resistance against any nucleic acid sequence or transcriptionproduct thereof but only to those sequences against which the CRISPRspacer (or pseudo CRISPR spacer) is homologous—such as 100% identical.

The CRISPR spacer(s) may be or may be derivable (preferably, derived)from an organism—such as a donor organism—that is different to thecell—such as the recipient cell or even a further donor organism—fromwhich the one or more cas genes or proteins and/or the one or more,preferably, two or more CRISPR repeats are or are derivable (preferably,derived). The CRISPR spacers may be or may be derivable (preferably,derived) from an organism—such as a donor organism—that is heterologousto the cell—such as the recipient cell or even a further donororganism—from which the one or more cas genes or proteins and/or the oneor more, preferably, two or more CRISPR repeats are or are derivable(preferably, derived). The one or more cas genes or proteins and/or theone or more, preferably, two or more CRISPR repeats may be or may bederivable (preferably, derived) from a homologous (ie. the same)cell—such as a homologous recipient cell.

For the avoidance of doubt, the CRISPR spacer(s) may be designed andproduced synthetically (eg. using recombinant DNA techniques).

In one embodiment, the CRISPR spacers are heterologous (ie. different)to the cell—such as the recipient cell—from which the one or more casgenes or proteins and/or the one or more, preferably, two or more CRISPRrepeats are or are derivable (preferably, derived) and the one or morecas genes or proteins and/or the one or more, preferably, two or moreCRISPR repeats are or are derivable (preferably, derived) from ahomologous cell—such as a homologous recipient cell.

In another embodiment, the CRISPR spacers are heterologous (ie.different) to the cell—such as the recipient cell—from which the one ormore cas genes or proteins are or are derivable (preferably, derived).

In another embodiment, the CRISPR spacers are heterologous to thecell—such as the recipient cell and the one or more cas genes orproteins and/or the one or more, preferably, two or more CRISPR repeatsare or are derivable (preferably, derived) from a homologous cell—suchas a homologous recipient cell.

In another embodiment, the CRISPR spacers are heterologous to thecell—such as the recipient cell—whereas the one or more cas genes orproteins and/or the one or more, preferably, two or more CRISPR repeatsis/are homologous to the cell—such as the recipient cell.

In another embodiment, the CRISPR spacers are heterologous to therecipient cell, whereas the recipient cell is homologous for the one ormore cas genes or proteins and/or the one or more, preferably, two ormore CRISPR repeats.

In another embodiment, the CRISPR spacer used in accordance with thepresent invention is one which is not naturally associated with theCRISPR repeat and/or cas genes and/or functional CRISPR repeat-cas genecombination. In other words, the CRISPR spacer in the recombinant CRISPRlocus according to the present invention is heterologous to the CRISPRrepeat and/or cas genes of the CRISPR locus.

One or more of CRISPR spacers may be used to engineer a cell—such as arecipient cell. In particular, one or more CRISPR spacers may be used toengineer a cell—such as a recipient cell—that in combination with one ormore cas genes or proteins and/or one or more, preferably, two or moreCRISPR repeats (preferably, one or more functional combination thereof)can be used to modulate the resistance of a cell against a targetnucleic acid or a transcription product thereof.

Suitably one or more of CRISPR spacers may be used to engineer acell—such as a recipient cell. In particular, one or more CRISPR spacersare used to engineer a cell—such as a recipient cell—that in combinationwith one or more cas genes or proteins can be used to modulate theresistance of a cell against a target nucleic acid or a transcriptionproduct thereof.

By way of example, the CRISPR spacers may be inserted into the DNA of acell (eg. a recipient cell)—such as plasmid DNA or genomic DNA of acell—using various methods that are well known in the art.

By way of further example, the CRISPR spacers may be used as a templateupon which to modify (eg. mutate) the DNA of a cell (eg. a recipientcell)—such as plasmid DNA or genomic DNA—such that CRISPR spacers arecreated in the DNA of the cell.

By way of further example, CRISPR spacers may be cloned into aconstruct, a plasmid or a vector and the like which are then transformedinto the cell, using methods such as those described herein.

In a further aspect, there is also provided a method for identifying aCRISPR spacer for use in modulating the resistance of a cell against atarget nucleic acid or a transcription product thereof comprising thesteps of: (i) preparing a cell comprising at least two CRISPR repeatsand at least one cas gene or protein; (ii) identifying at least oneCRISPR spacer in an organism—such as a donor organism; (iii) modifyingthe sequence of the CRISPR spacer of the cell such that it has homologyto the CRISPR spacer of the donor organism comprising the target nucleicacid; and (iv) determining if the cell modulates resistance against thetarget nucleic acid, wherein modulation of the resistance of the cellagainst the target nucleic acid or transcription product thereof isindicative that the CRISPR spacer modulates the resistance of the cellagainst the target nucleic acid.

The CRISPR spacers may comprise or consist of the nucleotide sequenceset forth any one or more of in any of SEQ ID Nos. 23-460 and/or SEQ IDNos. 522-665.

A CRISPR spacer is flanked by two CRISPR repeats. In other words, aCRISPR spacer has at least one CRISPR repeat on each side.

Without wishing to be bound by any particular theory, the further agiven CRISPR spacer is from the 5′ end of the CRISPR locus (comprisingthe cas gene(s) and/or the leader sequence), the lower the resistanceconferred by that CRISPR spacer may be. Accordingly, in one embodimentof the present invention it is preferred that one or more of the first100 CRISPR spacers from the 5′ end of the CRISPR locus (comprising thecas genes and/or the leader sequence) are modified, more preferably,that one or more of the first 50 CRISPR spacers from the 5′ end of theCRISPR locus (comprising the cas genes and/or the leader sequence) aremodified, more preferably, that one or more of the first 40 CRISPRspacers from the 5′ end of the CRISPR locus (comprising the cas genesand/or the leader sequence) are modified, more preferably, that one ormore of the first 30 CRISPR spacers from the 5′ end of the CRISPR locus(comprising the cas genes and/or the leader sequence) are modified, morepreferably, that one or more of the first 20 CRISPR spacers from the 5′end of the CRISPR locus (comprising the cas genes and/or the leadersequence) are modified, more preferably, that one or more of the first15 CRISPR spacers from the 5′ end of the CRISPR locus (comprising thecas genes and/or the leader sequence) are modified, more preferably,that one or more of the first 10 CRISPR spacers from the 5′ end of theCRISPR locus (comprising the cas genes and/or the leader sequence) aremodified.

As will be appreciated by the skilled person, different bacteria havedifferent numbers of CRISPR spacers.

CRISPR Spacer Core

For a specific CRISPR type within a microbial species, the CRISPR spaceris typically represented by a defined predominant length, but the sizemay vary. CRISPR types described to date have been found to contain apredominant spacer length of between about 20 bp and about 58 bp.

As used herein, the term “CRISPR spacer core” means the length of theshortest observed spacer within a CRISPR type. Thus, by way of example,within Streptococcus thermophilus CRISPR Type 1, the dominant spacerlength is 30 bp with a minority of spacers between 28 bp and 32 bp insize. So in this particular example (S. thermophilus CRISPR Type 1), theCRISPR spacer core is defined as a continuous stretch of 28 bp.

Suitably, the CRISPR spacer core is homologous to the target nucleicacid or a transcription product thereof or an identified sequence overthe length of the core sequence. Although homology can also beconsidered in terms of similarity, in the context of the presentinvention it is preferred to express homology in terms of sequenceidentity. A homologous sequence is taken to include a CRISPR spacercore, which may be at least 90% identical or at least 91, 92, 93, 94,95, 96, 97, 98 or 99% identical to the target nucleic acid sequence or atranscription product thereof or an identified sequence over the lengthof the core sequence.

Suitably, the CRISPR spacer core is 100% identical to the target nucleicacid sequence or a transcription product thereof or an identifiedsequence over the length of the core sequence.

Pseudo-CRISPR Spacer

As used herein, the term “pseudo-CRISPR spacer” refers to a nucleic acidsequence present in an organism (eg. a donor organism)—such as abacteriophage—which is preferably essential for function and/or survivaland/or replication and/or infectivity and the like, and which forms aCRISPR spacer sequence; and/or can be used to form or prepare a CRISPRspacer sequence which is complementary to or homologous to thepseudo-CRISPR spacer; and/or can be used to modulate resistance.

One or more of pseudo CRISPR spacers or CRISPR spacer(s) which is/arecomplementary or homologous to the one or more pseudo CRISPR spacer(s)may be used to engineer a cell—such as a recipient cell. In particular,one or more pseudo CRISPR spacers or CRISPR spacer(s) which is/arecomplementary or homologous to the one or more pseudo CRISPR spacer(s)may be used to engineer a cell—such as a recipient cell—that incombination with one or more cas genes or proteins and/or one or moreCRISPR repeats (eg, one or more functional combinations thereof) can beused to modulate the resistance of a cell against a target nucleic acidor a transcription product thereof.

One or more pseudo CRISPR spacers or CRISPR spacer(s) which is/arecomplementary or homologous to the one or more pseudo CRISPR spacer(s)may be used to engineer a cell—such as a recipient cell—that incombination with one or more cas genes or proteins can be used tomodulate the resistance of a cell against a target nucleic acid or atranscription product thereof.

By way of example, the pseudo CRISPR spacers or CRISPR spacer(s) whichis/are complementary or homologous to the one or more pseudo CRISPRspacer(s) may be inserted into the DNA of a cell (eg. a recipientcell)—such as plasmid DNA or genomic DNA of a cell—using various methodsthat are well known in the art.

By way of further example, the pseudo CRISPR spacers may be used as atemplate upon which to modify (eg. mutate) the DNA of a cell (eg. arecipient cell)—such as plasmid DNA or genomic DNA—such that CRISPRspacers are created in the DNA of the cell. By way of further example,pseudo CRISPR spacers or CRISPR spacer(s) which is/are complementary orhomologous to the one or more pseudo CRISPR spacer(s) may be cloned intoa construct, a plasmid or a vector and the like which are thentransformed into the cell, using methods such as those described herein.

Nucleic Acid Sequence

In a further aspect, there is provided a nucleic acid sequence (eg. arecombinant or an isolated nucleic acid sequence) consisting essentiallyof at least one cas gene or protein.

The nucleic acid sequence may be DNA or RNA of genomic, synthetic orrecombinant origin e.g. cDNA. The nucleotide sequence may bedouble-stranded or single-stranded whether representing the sense orantisense strand or combinations thereof. Recombinant nucleic acidsequences may be prepared by use of recombinant DNA techniques, asdescribed herein. The target nucleic acid sequence may be or may bederived from a gene.

As used herein, the term “consisting essentially of” in the context ofthe nucleic acid sequence refers to a nucleic acid sequence comprisingone or more cas genes and excluding at least one further component of aCRISPR locus—such as the CRISPR repeats, the CRISPR spacers and/or thecommon leader sequence.

Accordingly, in one embodiment, there is provided a nucleic acidsequence consisting essentially of at least one cas gene and at leasttwo CRISPR repeats.

In a further embodiment, there is provided a nucleic acid sequenceconsisting essentially of at least one cas gene and at least one CRISPRspacer.

In a further embodiment, there is provided a nucleic acid sequenceconsisting essentially of at least one cas gene, at least one CRISPRspacer and at least two CRISPR repeats.

In a further embodiment, there is provided a nucleic acid sequencecomprising at least one cas gene with the proviso that at least onefurther component of a CRISPR locus is absent, suitably, with theproviso that at least one further component of a natural CRISPR locus isabsent (eg. substantially absent).

In a further embodiment, there is provided a nucleic acid sequencecomprising at least one cas gene with the proviso that the CRISPRspacers of the CRISPR locus are absent, suitably, with the proviso thatCRISPR spacers of a natural CRISPR locus are absent (eg. substantiallyabsent).

In a further embodiment, there is provided a nucleic acid sequencecomprising at least one cas gene with the proviso that the CRISPRrepeats of the CRISPR locus are absent, suitably, with the proviso thatthe CRISPR repeats of a natural CRISPR, locus are absent.

In a further embodiment, there is provided a nucleic acid sequencecomprising at least one cas gene with the proviso that the common leadersequences of the CRISPR locus are absent, suitably, with the provisothat the common leader sequences of the natural CRISPR locus are absent.

In a further embodiment, there is provided a nucleic acid sequencecomprising at least one cas gene with the proviso that the CRISPRspacers and the CRISPR repeats of the CRISPR locus are absent, suitably,with the proviso that the CRISPR spacers and the CRISPR repeats of thenatural CRISPR locus are absent.

In a further embodiment, there is provided a nucleic acid sequencecomprising at least one cas gene with the proviso that the CRISPRspacers and the CRISPR repeats of the CRISPR locus are absent, suitably,with the proviso that the CRISPR spacers and the CRISPR repeats of thenatural CRISPR locus are absent.

In a further embodiment, there is provided a nucleic acid sequencecomprising at least one cas gene with the proviso that the CRISPRspacers and the common leader sequences of the CRISPR locus are absent,suitably, with the proviso that the CRISPR spacers and the common leadersequences of the natural CRISPR locus are absent.

In a further embodiment, there is provided a nucleic acid sequencecomprising at least one cas gene with the proviso that the CRISPRrepeats and the common leader sequences of the CRISPR locus are absent,suitably, with the proviso that the CRISPR repeats and the common leadersequences of the natural CRISPR locus are absent.

In further embodiment, there is provided a nucleic acid sequencecomprising at least one cas gene with the proviso that the CRISPRrepeats, the CRISPR spacers and the common leader sequences of theCRISPR locus are absent, suitably, with the proviso that the CRISPRrepeats, the CRISPR spacers and the common leader sequences of thenatural CRISPR locus are absent.

The nucleic acid sequence and the nucleic acids may be isolated orsubstantially purified. By “isolated” or “substantially purified” isintended that the nucleic acid molecules, or biologically activefragments or variants, homologues, or derivatives thereof aresubstantially or essentially free from components normally found inassociation with the nucleic acid in its natural state. Such componentsinclude other cellular material, culture media from recombinantproduction, and various chemicals used in chemically synthesising thenucleic acids.

An “isolated” nucleic acid sequence or nucleic acid is typically free ofnucleic acid sequences that flank the nucleic acid of interest in thegenomic DNA of the organism from which the nucleic acid was derived(such as coding sequences present at the 5′ or 3′ ends). However, themolecule may include some additional bases or moieties that do notdeleteriously affect the basic characteristics of the composition.

The nucleic acid sequence(s) may be used in the engineering of acell—such as a recipient cell. By way of example, the nucleic acidsequence may be inserted into the DNA—such as plasmid DNA or genomicDNA—of a recipient cell, using methods—such as homologous recombination.By way of further example, the nucleic acid sequence(s) may be used as atemplate upon which to modify (eg. mutate) the DNA of a cell (eg. arecipient cell)—such as plasmid DNA or genomic DNA—such that the nucleicacid sequence(s) are created in the DNA of the cell. By way of furtherexample, the nucleic acid sequence(s) may be cloned into a construct, aplasmid or a vector and the like which are then transformed into thecell, using methods such as those described herein.

A CRISPR spacer is flanked by two CRISPR repeats. In other words, aCRISPR spacer has at least one CRISPR repeat on each side.

Target Nucleic Acid Sequence

As used herein, the term “target nucleic acid sequence” refers to anynucleic acid sequence or transcription product thereof, against whichresistance in a cell—such as a recipient cell—is to be modulated.

The resistance may be against the target nucleic acid sequence per se.Advantageously, this confers resistance to a cell against a donororganism from which the target nucleic acid(s) is derivable (preferably,derived). Thus, by way of example, the insertion of a pseudo CRISPRspacer derivable (preferably, derived) from a bacteriophage or a CRISPRspacer(s) which is/are complementary or homologous to the one or morepseudo CRISPR spacer(s) into a cell—such as a recipient cell—may conferresistance to the bacteriophage. Thus, by way of further example,insertion between two CRISPR repeats of a pseudo CRISPR spacer derivable(preferably, derived) from a bacteriophage or CRISPR spacer(s) whichis/are complementary or homologous to the one or more pseudo CRISPRspacer(s) into a cell—such as a recipient cell—may confer resistance tothe bacteriophage.

The resistance may be against a transcription product of the targetnucleic acid sequence—such as a transcript of the target nucleic acidsequence (eg. an RNA (eg. mRNA) transcript (eg. a sense or an antisenseRNA transcript) or even a polypeptide transcription product.Advantageously, this confers resistance to a cell against a donororganism from which the transcription product is derivable (preferably,derived).

The target nucleotide sequence may be DNA or RNA of genomic, syntheticor recombinant origin.

The nucleotide sequence may be double-stranded or single-strandedwhether representing the sense or antisense strand or combinationsthereof.

The nucleotide sequence may be prepared by use of recombinant DNAtechniques (e.g. recombinant DNA).

The nucleotide sequence may be the same as a naturally occurring form,or may be derivable (preferably, derived) therefrom.

The target nucleic acid sequence may be or may be derivable (preferably,derived) from a gene.

The target nucleic acid sequence may be or may be derivable (preferably,derived) from a variant, homologue, fragment or derivative of a gene.

In one embodiment, the target nucleic sequence is or is derivable(preferably, derived) from bacteriophage.

In one embodiment, the target nucleic sequence is or is derivable(preferably, derived) from plasmid DNA.

In one embodiment, the target nucleic sequence is or is derivable(preferably, derived) from a mobile genetic element.

In one embodiment, the target nucleic sequence is or is derivable(preferably, derived) from a transposable element or an insertionsequence.

In one embodiment, the target nucleic sequence is or is derivable(preferably, derived) from a gene that confers resistance.

In one embodiment, the target nucleic sequence is or is derivable(preferably, derived) from a gene that confers resistance to anantibiotic.

In one embodiment, the target nucleic sequence is or is derivable(preferably, derived) from a virulence factor.

In one embodiment, the target nucleic sequence is or is derivable(preferably, derived) from a toxin, an internalin or a hemolysin.

Modulating Resistance

In a further aspect, there is provided a method for modulating theresistance of a cell—such as a recipient cell—against a target nucleicacid or a transcription product thereof.

As used herein, the term “modulating resistance” may refer tosuppressing, reducing, decreasing, inducing, conferring, restorating,elevating, increasing or otherwise affecting the resistance of a cell toa target nucleic acid.

As used herein, the term “resistance” is not meant to imply that a cellis 100% resistant to a target nucleic acid or a transcription productthereof, but includes cells that are tolerant of the target nucleic acidor a transcription product thereof.

As used herein the term “resistance to target nucleic acid ortranscription product thereof” means that resistance is conferredagainst a cell or an organism—such as a phage—that comprises or producesthe target nucleic acid or transcription product thereof.

Without being bound by any particular theory, we believe that resistanceor immunity is not linked to the “entry” of foreign DNA into a cell (ie.penetration through the cell membrane). Immunity or resistance wouldrather correspond to an obstruction, hurdle, impediment, barrier oravoidance to persistency, maintenance or survival of the incomingnucleic acid (either, for example, in a free linear form, or integratedwithin the bacterial chromosome, outside from a CRISPR locus or within acircular molecule—such as a plasmid), or to a obstruction, hurdle,impediment, barrier or avoidance to its replication and/or transcriptionand/or expression.

In one embodiment, the minimal components conferring immunity orresistance against a target nucleic acid or expression product thereofis at least one cas gene (or one Cas protein) and at least two CRISPRrepeats flanking a spacer.

In one embodiment, it is preferred that “modulating resistance” meansinducing, conferring, elevating or increasing the resistance of a cellto a target nucleic acid.

In one aspect, there is provided a method for modulating (e.g.conferring or increasing) the resistance of a cell against a targetnucleic acid or a transcription product thereof comprising the steps of:(i) identifying a sequence (eg. a conserved sequence) in an organism(preferably, a sequence essential to the function or survival of theorganism); (ii) preparing a CRISPR spacer which is a sequencehomologous, (suitably 100% identical), to the identified sequence; (iii)preparing a nucleic acid comprising at least one cas gene and at leasttwo CRISPR repeats together with the CRISPR spacer; and (iv)transforming a cell with said nucleic acid thus to render the cellresistant to said target nucleic acid or transcription product thereof.

As used herein, the term “conserved sequence” in the context ofidentifying a conserved sequence in an organism does not necessarilyhave to be conserved in its strictest sense since the knowledge of onesequence from a given organism will be enough. Furthermore the sequencedoes not need to be part of an essential entity, since we believe that aspacer inspired from an essential gene would be more efficient inconferring immunity or resistance.

In one embodiment, the conserved sequence is a sequence that isessential for function and/or survival and/or replication and/orinfectivity and the like of an organism or a cell. By way of example,the conserved sequence may be a helicase, a primase a head or tailstructural protein, a protein with a conserved domain (eg. holing,lysine, and others) or a conserved sequences amongst important phagegenes.

In a further aspect, there is provided a method for modulating (eg.conferring or increasing) the resistance of a cell against a targetnucleic acid or a transcription product thereof comprising the steps of:(i) identifying one or more CRISPR spacers in an organism resistant tothe target nucleic acid or transcription product thereof; (ii) preparinga recombinant nucleic acid comprising at least one cas gene or proteinand at least two CRISPR repeats together with said identified one ormore spacers; and (iii) transforming a cell with said recombinantnucleic acid thus to render the recipient cell resistant to said targetnucleic acid or transcription product thereof.

In a further aspect, there is provided a method for modulating (eg.conferring or increasing) the resistance of a cell comprising at leastone or more cas genes or proteins and one or more, preferably, two ormore CRISPR repeats against a target nucleic acid or a transcriptionproduct thereof comprising the steps of: (i) identifying one or moreCRISPR spacers in an organism resistant to the target nucleic acid ortranscription product thereof; and (ii) modifying the sequence of one ormore CRISPR spacer(s) in the cell such that the CRISPR spacer(s) hashomology to the CRISPR spacer(s) in the organism.

In one embodiment, one or more CRISPR spacers in a cell—such as arecipient cell—are modified (eg. genetically engineered) such that theCRISPR spacer(s) have homology to one or more CRISPR spacer(s) in anorganism—such as a donor organism—that is substantially resistant to atarget nucleic acid or a transcription product thereof in order torender the cell resistant to the target nucleic acid.

Suitably, the one or more cas genes or proteins and one or more,preferably, two or more CRISPR repeats in the cell are a functionalcombination as described herein.

The genetic engineering may include, but is not limited to, adding (eg.inserting), deleting (eg. removing) or modifying (eg. mutating) thesequence of the one or more CRISPR spacers or in a cell such that theCRISPR spacer has homology (eg. increased homology after the geneticengineering) to one or more CRISPR spacers of a donor organism. Thisengineering step will result in a cell that was substantially sensitiveto a target nucleic acid or a transcription product thereof beingsubstantially resistant to the target nucleic acid or a transcriptionproduct thereof.

The genetic engineering may even include, but is not limited to, adding(eg. inserting) or deleting (eg. removing) the sequence of the one ormore pseudo CRISPR spacers in to a cell. This engineering step willresult in a cell that was substantially sensitive to a target nucleicacid or a transcription product thereof being substantially resistant tothe target nucleic acid or a transcription product thereof.

In another embodiment, “modulating resistance” means suppressing,reducing or decreasing the resistance of a cell to a target nucleicacid.

Thus, in a further aspect, there is provided a method for decreasing orreducing the resistance of a cell—such as a recipient cell—comprising atleast one or more cas genes or proteins and one or more, preferably, twoor more CRISPR repeats against a target nucleic acid or a transcriptionproduct thereof.

According to this embodiment, the method comprises the steps of: (i)identifying one or more CRISPR spacers in an organism that issubstantially resistant to the target nucleic acid or a transcriptionproduct thereof; and (ii) modifying the sequence of one or more CRISPRspacer(s) in the cell such that the CRISPR spacer(s) has a reduceddegree of homology to the CRISPR spacer(s) in the organism.

In another embodiment, there is provided a method for modulating (eg.decreasing) the resistance of a cell comprising one or more cas genes orproteins and one or more, preferably, two or more CRISPR repeats againsta target nucleic acid or transcription product thereof comprising thesteps of: (i) identifying a CRISPR spacer or a pseudo CRISPR spacer inan organism comprising a target nucleic acid or transcription productthereof against which resistance is to be modulated; and (ii)identifying the CRISPR spacer in the organism in which resistance is tobe modulated; and (iii) adapting the sequence of the CRISPR spacer inthe organism in which resistance is to be modulated such that the CRISPRspacer has a lower degree of homology to the CRISPR spacer or pseudoCRISPR spacer of the organism comprising the target nucleic acid ortranscription product thereof against which resistance is to bemodulated.

One or more CRISPR spacers in a substantially resistant cell areengineered in order to render the cell sensitive to a target nucleicacid. The genetic engineering may include, but is not limited to, theaddition (eg. insertion), deletion (eg. removal) or modification of oneor more functional CRISPR repeat-cas combinations or portions orfragments thereof in the substantially resistant cell and/or theaddition (eg. insertion), deletion (eg. removal) or modification of oneor more CRISPR spacers or portions or fragments thereof in thesubstantially resistant cell.

This engineering step will then result in a cell that was substantiallyresistant to a target nucleic acid or a transcription product thereofbecoming substantially sensitive to a target nucleic acid or atranscription product thereof.

Typically, in order to confer sensitivity to a cell, it is expected thatone or more CRISPR spacers, one or more cas genes or proteins, one ormore, preferably, two or more CRISPR repeats or one or more functionalCRISPR repeat-cas combinations from a substantially resistant cell willbe removed, deleted or modified such that resistance is no longerconferred.

Advantageously, cells that are sensitive to a target nucleic acid or atranscription product thereof may be prepared such that their levelswithin a given culture—such as a starter culture—may be modulated (eg.decreased) as desired. Thus, by way of example, a starter culturecomprising two or more bacterial strains may be developed such that allmembers of the culture are sensitive to the same agent (eg.bacteriophage). Accordingly, at a time when it is no longer desired forthe culture to be alive, the culture may be contacted with the samesingle agent in order to kill all members of the culture.

Moreover, it may even be possible to modulate the sensitivity of a cellto one or more agents (eg. bacteriophage) such that the agent kills onlya certain proportion of the cells in a given culture—such as 10, 20, 30,40, 50, 60, 70, 80, 90 or 95% of the cells in a given culture.

In one aspect, a cell—such as a recipient cell—may be engineered suchthat it comprises a CRISPR spacer or a sequence corresponding to apseudo CRISPR spacer thereby rendering the cell resistant to a targetnucleic acid or transcription product thereof. Suitably, the cell isengineered such that the CRISPR spacer or sequence corresponding to thepseudo CRISPR spacer is used together with a functional cas gene-CRISPRrepeat combination, as described herein.

In one aspect, a cell that is resistant to a target nucleic acid ortranscription product thereof is engineered such that the CRISPR spacerconferring the immunity against the target nucleic acid or transcriptionproduct thereof is inserted into a cell that comprises a functional casgene-CRISPR repeat combination, thereby rendering the cell resistant tothe target nucleic acid or transcription product thereof.

In one aspect, the sequence of one or more CRISPR spacers or pseudoCRISPR spacers of a cell that is resistant to a target nucleic acid ortranscription product thereof is determined. A cell—such as a recipientcell—is then engineered such that it comprises the sequence of theCRISPR spacer and a functional cas gene-CRISPR repeat combination,thereby rendering the cell resistant to the target nucleic acid ortranscription product thereof.

In one aspect, a CRISPR spacer from a cell—such as a recipient cell—anda functional cas gene-CRISPR repeat combination from the same ordifferent cell—such as the same or different recipient cell—areprepared. A further cell—such as a recipient cell—is then engineeredsuch that is comprises the CRISPR spacer sequence and functional casgene-CRISPR repeat combination thereby rendering the cell resistant tothe target nucleic acid or transcription product thereof.

A CRISPR spacer is flanked by two CRISPR repeats. In other words, aCRISPR spacer has at least one CRISPR repeat on each side.

Bacteriophage

In a particularly preferred aspect of the present invention, theresistance of a cell against a bacteriophage is modulated.

The bacteriophage is virulent to the cell.

The bacteriophage may be a virulent or a temperate bacteriophage.

As used herein, the term “bacteriophage” has its conventional meaning asunderstood in the art ie. a virus that selectively infectsprokaryotes—such as bacteria. Many bacteriophages are specific to aparticular genus or species or strain of cell.

The bacteriophage may be a lytic bacteriophage or a lysogenicbacteriophage.

A lytic bacteriophage is one that follows the lytic pathway throughcompletion of the lytic cycle, rather than entering the lysogenicpathway. A lytic bacteriophage undergoes viral replication leading tolysis of the cell membrane, destruction of the cell, and release ofprogeny bacteriophage particles capable of infecting other cells.

A lysogenic bacteriophage is one capable of entering the lysogenicpathway, in which the bacteriophage becomes a dormant, passive part ofthe cell's genome through prior to completion of its lytic cycle.

The term “bacteriophage” is synonymous with the term “phage”.

Whilst resistance against any bacteriophage (including wild type,naturally occurring, isolated or recombinant bacteriophage) may beemployed, bacteriophage active against bacteria are preferred. Moresuitably, bacteriophage active against bacteria that are pathogenic toplants and/or animals (including humans) are of particular interest.

By way of example, the bacteriophage include, but are not limited to,those bacteriophage capable of infecting a bacterium that naturallycomprises one or more CRISPR loci. CRISPR loci have been identified inmore than 40 prokaryotes (Jansen et al. 2002b; Mojica et al., 2005; Haftet al., 2005) including, but not limited to Aeropyrum, Pyrobaculum,Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus,Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma,Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas,Chlorobium, Thermus, Bacillus, Listerta, Staphylococcus, Clostridium,Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium,Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus,Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia,Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella,Xanthamonas, Yersinia, Treponema and Thermotoga.

By way of example, the bacteriophage include, but are not limited to,those bacteriophage capable of infecting bacteria belonging to thefollowing. genera: Escherichia, Shigella, Salmonella, Erwinia, Yersinia,Bacillus, Vibrio, Legionella, Pseudomonas, Neisseria, Bordetella,Helicobacter, Listeria, Agrobacterium, Staphylococcus, Streptococcus,Enterococcus, Clostridium, Corynebacterium, Mycobacterium, Treponema,Borrelia, Francisella, Brucella and Xanthomonas.

By way of further example, the bacteriophage include, but are notlimited to, those bacteriophage capable of infecting (or transducing)lactic acid bacteria species, a Bifidobacterium species, aBrevibacterium species, a Propionibacterium species, a Lactococcusspecies, a Streptococcus species, a Lactobacillus species including theLactobacillus acidophilus, Enterococcus species, Pediococcus species, aLeuconostoc species and Oenococcus species.

By way of further example, the bacteriophage include, but are notlimited to, those bacteriophage capable of infecting Lactococcus lactis,including Lactococcus lactis subsp. lactis and Lactococcus lactis subsp.cremoris, Lactococcus lactis subsp. lactis biovar diacetylactfis,Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus,Lactobacillus helveticus, Bifidobacterium lactis, Lactobacillusacidophilus, Lactobacillus casei, Bifidobacterium infantis,Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillusplantarum, Lactobacillus reuteri, Lactobacillus gasseri, Lactobacillusjohnsonii or Bifidobacterium longum.

By way of further example, the bacteriophages include, but are notlimited to, those bacteriophage capable of infecting any fermentativebacteria susceptible to disruption by bacteriophage infection, includingbut not limited to processes for the production of antibiotics, aminoacids, and solvents. Products produced by fermentation which are knownto have encountered bacteriophage infection, and the correspondinginfected fermentation bacteria, include Cheddar and cottage cheese(Lactucoccus lactis subsp. lactis, Lactococcus lactis subsp. cremoris),Yogurt (Lactobacillus delbrueckii subsp. bulgaricus, Streptococcusthermophilus), Swiss cheese (S. thermophilus, Lactobacillus lactis,Lactobacillus helveticus), Blue cheese (Leuconostoc cremoris), Italiancheese (L. bulgaricus, S. thermophilus), Viili (Lactococcus lactissubsp. cremoris, Lactococcus lactis subsp. lactis biovar diacetylactis,Leuconostoc cremoris), Yakult (lactobacillus casei), casein (Lactococcuslactis subsp. cremoris), Natto (Bacillus subtilis var. natto), Wine(Leuconostoc oenos), Sake (Leuconostoc mesenteroides), Polymyxin(Bacillus polymyxa), Colistin (Bacillus colistrium), Bacitracin(Bacillus licheniformis), L-Glutamic acid (Brevibacteriumlactofermentum, Microbacterium ammoniaphilum), and acetone and butanol(Colstridium acetobutylicum, Clostridium saccharoperbutylacetonicum).

Preferred bacteria are S. thermophilus, L. delbrueckii subsp. bulgaricusand/or L. acidophilus.

By way of further example, the bacteriophages include, but are notlimited to, those bacteriophage capable of infecting a bacterium thatcomprises one or more heterologous CRISPR loci. The bacterium maycomprise one or more heterologous CRISPR loci, one or more heterologouscas genes, one or more heterologous CRISPR repeats and/or one or moreheterologous CRISPR spacers.

Bacteriophages may include, but are not limited to, bacteriophages thatbelong to any of the following virus families: Corticoviridae,Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae,Podoviridae, Siphoviridae, or Tectiviridae.

To cause bacteriophage infection of cells, it “infects” a cell when itinjects or transfers its nucleic acid into the cell, with the phagenucleic acid existing independently of the cell's genome. Infection maylead to expression (transcription and translation) of the bacteriophagenucleic acid within the cell and continuation of the bacteriophage lifecycle. In the case of recombinant bacteriophage, recombinant sequenceswithin the phage genome, such as reporter nucleic acids, may beexpressed as well.

It has been found that CRISPR spacer sequences in prokaryotes often havesignificant similarities to a variety of DNA molecules—such as geneticelements (including, but not limited to, chromosomes, bacteriophages,conjugative plasmids). Interestingly, cells carrying these CRISPRspacers are unable to be infected by DNA molecules containing sequenceshomologous to the spacers (Mojica et al. 2005).

In the context of the present invention, one or more particularpseudo-spacers derivable or derived from bacteriophage DNA or CRISPRspacer(s) which is/are complementary or homologous to the one or morepseudo-CRISPR spacer(s) can be added within a CRISPR locus of acell—such as a recipient cell—in order to modulate (eg. provide)resistance against a particular bacteriophage, thus substantiallypreventing phage attack.

Typically, particular regions within the phage genome may be targeted toprepare the pseudo-spacers—such as genes coding for host specificityproteins—that provide particular phage-host recognition—such ashelicases, primase, head or tail structural proteins, proteins with aconserved domain (eg. holing, lysine, and others) or conserved sequencesamongst important phage genes.

Any nucleic acid originating from the phage genome may confer immunityagainst the phage when inserted, for example, between two repeats in anactive CRISPR locus. Immunity may be more “efficient” if the CRISPRspacer corresponds to an internal sequence of a phage gene, and evenmore “efficient” when this gene encodes “essential” proteins (eg. theantireceptor).

Accordingly, in a further aspect, there is provided a method forconferring resistance to a cell (suitably, a bacterial cell) against abacteriophage comprising the steps of: (a) providing one or more pseudoCRISPR. spacers from at least one bacteriophage; (b) identifying one ormore functional CRISPR repeat-cas combinations in at least one cell thatis substantially sensitive to the bacteriophage; and (c) engineering theone or more CRISPR loci in the substantially sensitive cell such thatthey comprise one or more pseudo CRISPR spacers from a bacteriophage orone or more CRISPR spacer(s) which is/are complementary or homologous tothe one or more pseudo CRISPR spacer(s) to render the cell resistant.

In a further aspect, there is provided a method for conferringresistance to a cell (suitably, a bacterial cell) against abacteriophage comprising the steps of (a) providing one or more pseudoCRISPR spacers from at least one bacteriophage; (b) identifying one ormore functional CRISPR repeat-cas combinations in at least one cell thatis substantially sensitive to the bacteriophage; and (c) inserting oneor more pseudo CRISPR spacers from the bacteriophage or one or moreCRISPR spacer(s) which is/are complementary or homologous to the one ormore pseudo CRISPR spacer(s) into the substantially sensitive cell suchthat the cell is rendered substantially resistant to the bacteriophage.

In a further aspect, there is provided a method for modulating thelysotype of a bacterial cell comprising the steps of: (a) providing oneor more pseudo CRISPR spacers from, at least one bacteriophage; (b)identifying one or more functional CRISPR repeat-cas combinations in atleast one cell that is substantially sensitive to the bacteriophage; and(c) engineering the one or more CRISPR loci in the substantiallysensitive cell such that they comprise one or more pseudo CRISPR spacersfrom a bacteriophage or one or more CRISPR spacer(s) which is/arecomplementary or homologous to the one or more pseudo CRISPR spacer(s).

In a further aspect, there is provided a method for modulating thelysotype of a bacterial cell comprising the steps of: (a) providing oneor more pseudo CRISPR spacers from at least one bacteriophage; (b)identifying one or more functional CRISPR repeat-cas combinations in atleast one cell that is substantially sensitive to the bacteriophage; and(c) inserting one or more one or more pseudo CRISPR spacers from thebacteriophage or one or more CRISPR spacer(s) which is/are complementaryor homologous to the one or more pseudo CRISPR spacer(s) into thesubstantially sensitive cell.

In a further aspect, there is provided a method for conferringresistance to a cell (suitably, a bacterial cell) against abacteriophage comprising the steps of: (i) identifying a pseudo CRISPRspacer in a bacteriophage comprising a target nucleic acid or atranscription product thereof against which resistance is to bemodulated; and (ii) modifying the sequence of the CRISPR spacer of thecell such that the CRISPR spacer of the cell has homology to the pseudoCRISPR spacer of the bacteriophage comprising the target nucleic acid.

In a further aspect, there is provided a method for conferringresistance to a cell (suitably, a bacterial cell) against abacteriophage comprising the steps of: (i) identifying a pseudo CRISPRspacer in a bacteriophage comprising a target nucleic acid or atranscription product thereof against which resistance is to bemodulated; and (ii) modifying the sequence of the CRISPR spacer of thecell such that the CRISPR spacer of the cell has 100% homology oridentity to the pseudo CRISPR spacer of the bacteriophage comprising thetarget nucleic acid.

In a further aspect, there is provided a method for modulating thelysotype of a bacterial cell comprising the steps of: comprising thesteps of: (i) identifying a pseudo CRISPR spacer in a bacteriophagecomprising a target nucleic acid or a transcription product thereofagainst which resistance is to be modulated; and (ii) modifying thesequence of the CRISPR spacer of the cell such that the CRISPR spacer ofthe cell has homology to the pseudo CRISPR spacer of the bacteriophagecomprising the target nucleic acid.

In a further aspect, there is provided a method for modulating thelysotype of a bacterial cell comprising the steps of: (i) identifying apseudo CRISPR spacer in a bacteriophage comprising a target nucleic acidor a transcription product thereof against which resistance is to bemodulated; and (ii) modifying the sequence of the CRISPR spacer of thecell such that the CRISPR spacer of the cell has 100% homology oridentity to the pseudo CRISPR spacer of the bacteriophage comprising thetarget nucleic acid:

Suitably, the CRISPR spacer of the bacterial cell will have 100%homology or identity to a sequence—such as a pseudo CRISPR spacer—in thebacteriophage comprising the target nucleic acid.

Suitably, the CRISPR spacer of the bacterial cell will form a componentpart of a CRISPR locus comprising a functional CRISPR repeat-cascombination as described herein.

Suitably, the target nucleic acid or a transcription product thereof inthe bacteriophage is a highly conserved nucleic acid sequence.

Suitably, the target nucleic acid or transcription product thereof inthe bacteriophage is a gene coding for a host specificity protein.

Suitably, the target nucleic acid or transcription product thereof inthe bacteriophage encodes an enzyme that is essential for survival,replication or growth of the bacteriophage.

Suitably, the target nucleic acid or transcription product thereof inthe bacteriophage encodes a helicase, a primase, a head or tailstructural protein, or a protein with a conserved domain (eg. holing,lysine, and others).

Advantageously, bacterial cells may be prepared according to the presentinvention that have a “reduced susceptibility to bacteriophagemultiplication or infection”. As used herein, this term refers to thebacterium as having a low or no susceptibility to bacteriophagemultiplication or infection when compared to the wild-type bacteriumwhen cultured, in for example, a dairy medium.

In one embodiment, the term “low susceptibility to bacteriophagemultiplication” refers to the level of bacteriophage multiplication in abacterium being below a level, which would cause a deleterious effect toa culture in a given period of time. Such deleterious effects on aculture include, but are not limited to, no coagulation of milk duringproduction of fermented milk products (such as yoghurt or cheese),inadequate or slow lowering of the pH during production of fermentedmilk products (such as yoghurt or cheese), slow ripening of cheese anddeterioration of a food's texture to the point where it is unappetisingor unsuitable for human consumption.

For an equivalent set of culture conditions the susceptibility towards abacteriophage of a bacterium of the present invention is, in comparisonto the wild-type bacterium, 100 times lower (efficiency of plaquing[EOP]=10⁻²), preferably 1000 times lower (EOP=10⁻³), preferably 10 000times lower (EOP=10⁻⁴), more preferably 100 000 times lower (EOP=10⁻⁵).Preferably, the level of bacteriophage multiplication in a culture ismeasured after about 14 hours incubation of the culture, more preferablyafter about 12 hours, more preferably after about 7 hours, morepreferably after about 6 hours, more preferably after about 5 hours andmore preferably after about 4 hours.

In a further aspect, there is provided a method for conferringsensitivity to a cell (preferably, a bacterial cell) against abacteriophage comprising the steps of: (a) providing a pseudo CRISPRspacer from at least one bacteriophage; (b) identifying one or morefunctional CRISPR repeat-cas combinations in a cell that issubstantially resistant to the bacteriophage; and (c) engineering theone or more CRISPR loci in the substantially, sensitive cell such thatthey comprise one or more pseudo CRISPR spacers or one or more CRISPRspacer(s) which is/are complementary or homologous to the one or morepseudo CRISPR spacer(s) that have a reduced degree of homology ascompared to the one or more CRISPR loci in the substantially resistantcell.

In a further aspect, there is provided a method for modulating (eg.reducing) the lysotype of a cell (preferably a bacterial cell),comprising one or more cas genes or proteins and one or more,preferably, two or more CRISPR repeats comprising the steps of: (i)identifying a pseudo CRISPR spacer in a bacteriophage against whichresistance is to be modulated; and (ii) modifying the sequence of theCRISPR spacer of the cell such that the CRISPR spacer of the cell has areduced degree of homology to the pseudo CRISPR spacer of thebacteriophage comprising the target nucleic acid.

In still a further aspect, there is provided a method for modulating(eg. reducing or decreasing) the resistance of a bacterial cellcomprising one or more cas genes or proteins and one or more,preferably, two or more CRISPR repeats against a bacteriophagecomprising the steps of: (i) identifying one or more pseudo CRISPRspacers in a bacteriophage against which resistance is to be modulated;(ii) identifying a CRISPR spacer in the bacterial cell in whichresistance is to be modulated that is homologous to the pseudo CRISPRspacer(s); and (iii) modifying the sequence of the CRISPR spacer in thebacterial cell in which resistance is to be modulated such that theCRISPR spacer has a lower degree of homology to the pseudo CRISPRspacer(s) of the bacteriophage against which resistance is to bemodulated.

Suitably, the CRISPR spacer of the cell will have a reduced degree ofhomology—such as a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 90 or 95% reduction in homology ascompared to the pseudo CRISPR spacer(s) of the bacteriophage againstwhich resistance is to be modulated.

Bacterial cells may therefore be prepared according to the presentinvention that have an “increased susceptibility to bacteriophagemultiplication”. As used herein, this term refers to the bacterium ashaving an increased or high susceptibility to bacteriophagemultiplication when compared to the wild-type bacterium when cultured,in for example, a dairy medium.

In one embodiment, the term “high susceptibility to bacteriophagemultiplication” refers to the level of bacteriophage multiplication in abacterium being above a level, which would cause a deleterious effect toa culture in a given period of time. Such deleterious effects on aculture include, but are not limited to, no coagulation of milk duringproduction of fermented milk products (such as yoghurt or cheese),inadequate or slow lowering of the pH during production of fermentedmilk products (such as yoghurt or cheese), slow ripening of cheese anddeterioration of a food's texture to the point where it is unappetisingor unsuitable for human consumption. For an equivalent set of cultureconditions the susceptibility towards a bacteriophage of a bacterium ofthe present invention is, in comparison to the wild-type bacterium, 100times higher, 1000 times higher, 10 000 times higher, or 100 000 timeshigher (EOP=10⁻⁵). The level of bacteriophage multiplication in aculture. is measured after about 14 hours incubation of the culture,more preferably after about 12 hours, more preferably after about 7hours, more preferably after about 6 hours, more preferably after about5 hours and in a highly preferred embodiment after about 4 hours.

A CRISPR spacer is flanked by two CRISPR repeats. In other words, aCRISPR spacer has at least one CRISPR repeat on each side.

Bacteria

In a further embodiment, the target nucleic sequence or a transcriptionproduct thereof may be or may be derivable (preferably, derived) fromone or more bacteria. Accordingly, resistance of a cell, eg. a bacterialcell, against bacteria or a component thereof may be modulated.

The target nucleotide sequence may be or may be derived from a gene thatis or is associated with resistance to plasmid transfer in bacteria.According to this embodiment of the present invention, one or moreCRISPR spacers in the cell are modified such that the CRISPR spacer ofthe cell has homology to the CRISPR spacer and/or pseudo CRISPR spacercontained in the plasmid DNA of the bacterial cell so as to provideresistance against the particular plasmid(s), thus preventing transferof foreign DNA into the cell. Specifically, particular regions withinthe plasmid DNA can be targeted as to provide immunity against plasmidDNA, such as sequences within the plasmids origin of replication orsequences within genes coding for replication proteins.

Thus, according to this aspect, the method comprises the steps of: (i)identifying a CRISPR spacer and/or pseudo CRISPR spacer derivable(preferably, derived) from the plasmid DNA of a bacterial cell againstwhich resistance is to be modulated; and (ii) modifying the sequence ofa CRISPR spacer in the cell in which resistance is to be modulated suchthat the CRISPR spacer of the cell has homology to the CRISPR spacerand/or pseudo CRISPR spacer contained in the plasmid DNA of thebacterial cell.

In still a further aspect, there is provided a further method forconferring resistance to a cell against plasmid transfer comprising thesteps of: (a) identifying a CRISPR spacer and/or pseudo CRISPR spacerderivable (preferably, derived) from plasmid DNA; (b) identifying one ormore functional CRISPR repeat-cas gene combinations in a cell that issubstantially sensitive to the plasmid; and (c) engineering the one ormore CRISPR loci in the substantially sensitive cell such that theycomprise one or more CRISPR spacers and/or pseudo CRISPR spacers fromthe plasmid to render the cell resistant.

The target nucleotide sequence may be or may be derived from a gene thatis or is associated with resistance to one or more mobile geneticelements. Particular CRISPR spacers and/or pseudo CRISPR spacersderivable (preferably, derived) from one or more mobile genetic elementscan be added within a CRISPR locus of a cell so as to provide resistanceagainst mobile genetic elements—such as transposable elements andinsertion sequences, thus preventing transfer of foreign DNA and geneticdrift. Specifically, particular regions within transposons and insertionsequences can be targeted so as to provide immunity against mobilegenetic elements. For example, targets can include conjugativetransposons (Tn916), class II transposons (Tn501), insertions sequences(IS26) or transposase genes.

Thus, according to this aspect, the method comprises the steps of: (i)identifying a CRISPR spacer and/or pseudo CRISPR spacer derivable(preferably, derived) from one or more mobile genetic elements of a cellagainst which resistance is to be modulated; and (ii) modifying thesequence of a CRISPR spacer in a cell in which resistance is to bemodulated such that the CRISPR spacer and/or pseudo CRISPR spacer of thecell has homology to the CRISPR spacer contained in the mobile geneticelement(s) of the cell.

In still a further aspect, there is provided a further method forconferring resistance to a cell against one or more mobile geneticelements comprising the steps of: (a) identifying a CRISPR spacer and/orpseudo CRISPR spacer derivable (preferably, derived) from one or moremobile genetic elements; (b) identifying one or more functional CRISPRrepeat-cas combinations in a cell that is substantially sensitive to theone or more mobile genetic elements; and (c) engineering the one or moreCRISPR loci in the substantially sensitive cell such that they compriseor have homology to one or more CRISPR spacers and/or pseudo CRISPRspacers from the one or more mobile genetic elements to render the cellresistant.

The target nucleotide sequence may be or may be derived from a gene thatis or is associated with resistance to antibiotics. By “antibiotic” isunderstood a chemical composition or moiety which decreases theviability or which inhibits the growth or reproduction of microbes.Antibiotic resistance genes include, but are not limited to bla_(tem),bla_(rob), bla_(shv), aadB, aacC1, aacC2, aacC3, aacA4, mecA, vanA,vanH, vanX satA, aacA-aphH, vat, vga, msrA sul, and/or int. Theantibiotic resistance genes include those that are or are derivable(preferably, derived) from bacterial species that include but are notlimited to the genera Escherichia, Klebsiella, Pseudomonas, Proteus,Streptococcus, Staphylococcus, Enterococcus, Haemophilus and Moraxella.The antibiotic resistance genes also include those that are or arederivable (preferably, derived) from bacterial species that include butare not limited to Escherichia coli, Klebsiella pneumoniae, Pseudomonasaeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcusaureus, Staphylococcus epidermidis, Enterococcus faecalis,Staphylococcus saprophyticus, Streptococcus pyogenes, Haemophilusinfluenzae, and Moraxella catarrhalis.

Particular CRISPR spacers and/or pseudo CRISPR spacers derivable(preferably, derived) from antibiotic resistance encoding genes can beadded within a CRISPR locus of a cell—such as a recipient cell—so as toprevent transfer of genes conferring resistance to antibiotics into thecell, thus reducing the risk of acquiring antibiotic resistance markers.By way of example, targets can also include vanR, (a gene conferringresistance to vancomycin), or tetR, a gene conferring resistance totetracycline, or targeting beta-lactamase inhibitors.

Thus, according to this aspect, the method comprises the steps of: (i)identifying one or more CRISPR spacers and/or pseudo CRISPR spacersderivable (preferably, derived) from a cell that comprises one or moreantibiotic resistance genes or markers; and (ii) modifying the sequenceof the CRISPR spacer in a cell that does not comprise or does notexpress the antibiotic resistance genes or markers such that the CRISPRspacer of the cell has homology to the one or more CRISPR spacers and/orpseudo CRISPR spacers contained in the cell that comprises one or moreantibiotic resistance genes or markers.

In still a further aspect, there is provided a method for modulating theacquisition of antibiotic resistance markers in a cell comprising thesteps of: (a) identifying one or more CRISPR spacers and/or pseudo.CRISPR spacers derivable (preferably, derived) from a cell thatcomprises one or more antibiotic resistance genes or markers; (b)identifying one or more CRISPR loci in a cell that does not comprise ordoes not express the antibiotic resistance genes or markers; and (c)modifying the sequence of the CRISPR spacer in the cell that does notcomprise or does not express the antibiotic resistance genes or markerssuch that the CRISPR spacer and/or pseudo CRISPR spacers has homology tothe CRISPR spacer contained in the cell resistant to the transfer ofgenes conferring resistance to one or more antibiotics.

The target nucleotide sequence may be or may be derived from a gene thatis or is associated with genes encoding virulence factors. ParticularCRISPR spacers and/or pseudo CRISPR spacers derivable (preferably,derived) from genes encoding virulence factors can be added within abacterium CRISPR locus to provide resistance against the transfer ofgenes conferring virulence into the bacterium. For example, factorscommonly contributing to virulence in microbial pathogens can betargeted, such as toxins, internalins and hemolysins.

Thus, according to this aspect, the method comprises the steps of: (i)identifying one or more CRISPR spacers and/or pseudo CRISPR spacersderivable (preferably, derived) from a cell that comprises one or morevirulence factors; and (ii) modifying the sequence of the CRISPR spacerin a cell that does not comprise or does not express the virulencefactor(s) or marker(s) such that the CRISPR spacer of the cell hashomology to the one or more CRISPR spacers and/or pseudo CRISPR spacerscontained in the cell that comprises one or more virulence factors.

In still a further aspect, there is provided a further method forconferring resistance to a cell against one or more virulence factor(s)or marker(s) comprising the steps of: (a) identifying a CRISPR spacerand/or pseudo CRISPR spacer derivable (preferably, derived) from one ormore virulence factor(s) or marker(s); (b) identifying one or morefunctional CRISPR repeat-cas combinations in a cell that issubstantially sensitive to the one or more virulence factor(s) ormarker(s); and (c) engineering the one or more CRISPR loci in thesubstantially sensitive cell such that they comprise one or more CRISPRspacers and/or pseudo CRISPR spacers from the one or more virulencefactor(s) or marker(s) to render the cell resistant.

A CRISPR spacer is flanked by two CRISPR repeats. In other words, aCRISPR spacer has at least one CRISPR repeat on each side.

Modification

Nucleic acid sequences may be modified by genetically engineeringnucleic acid sequences.

All or part of a nucleic acid sequence may be modified.

All or part of one or more CRISPR spacers, cas genes or proteins, CRISPRrepeats or CRISPR loci may be modified.

Recombinant CRISPR spacers, cas genes or proteins, CRISPR repeats orCRISPR loci may be modified.

Naturally occurring CRISPR spacers, cas genes or proteins, CRISPRrepeats or CRISPR loci may be modified.

Naturally co-occurring cas genes or proteins and CRISPR repeats may bemodified.

The genetic engineering may be mediated using various methods that areknown in the art and will typically include well known methods—such asPCR amplification, cloning and site-directed mutagenesis. Mutations maybe introduced using synthetic oligonucleotides. These oligonucleotidescontain nucleotide sequences flanking the desired mutation sites. Asuitable method is disclosed in Morinaga et al., (Biotechnology (1984)2, p646-649). Another method of introducing mutations intoenzyme-encoding nucleotide sequences is described in Nelson and Long(Analytical Biochemistry (1989), 180, p 147-151). A further method isdescribed in Sarkar and Sommer (Biotechniques (1990), 8, p404-407—“Themegaprimer method of site directed mutagenesis”). Commercially availablekits are also now widely available for performing site directedmutagenesis. Genetic engineering methods are described in detail in J.Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: ALaboratory Manual, Second Edition, Books 1-3, Cold Spring HarborLaboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements;Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley &Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNAIsolation and Sequencing: Essential Techniques, John Wiley & Sons; M. J.Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach,Irl Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods ofEnzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNAMethods in Enzymology, Academic Press.

The genetic engineering step may even include methods such as homologousrecombination which may be particularly useful when, for example, CRISPRspacers are being inserted or deleted.

The genetic engineering step may even include the activation of one ormore nucleic acid sequences—such as one CRISPR loci, CRISPR repeats,CRISPR spacers, cas genes or proteins, functional combinations of casgenes or proteins and CRISPR repeats or even combinations thereof.

Suitably, one or more CRISPR spacers or pseudo CRISPR spacers may beinserted into at least one CRISPR locus.

In one embodiment, the modification does not interrupt one or more casgenes of the at least one CRISPR locus. In another embodiment, the oneor more cas genes remain intact.

In one embodiment, the modification does not interrupt one or moreCRISPR repeats of the at least one CRISPR locus. In one embodiment, theone or more CRISPR repeats remain intact.

Suitably, one or more CRISPR spacers or pseudo CRISPR spacers may beinserted into or within at least one CRISPR locus.

Suitably, one or more CRISPR spacers or pseudo CRISPR spacers, may beinserted at the 5′ end of at least one CRISPR locus.

In one embodiment, the modification comprises inserting at least oneCRISPR spacer or pseudo CRISPR spacers into a cell—such as a recipientcell. In another embodiment, the modification comprises inserting one ormore CRISPR spacers or pseudo CRISPR spacers into (eg. to modify orreplace) one or more CRISPR spacers of a cell—such as a recipient cell.

In one embodiment, the modification comprises inserting at least oneCRISPR spacer or pseudo CRISPR spacer from an organism—such as a donororganism—into the cell. In another embodiment, the modificationcomprises inserting one or more CRISPR spacers or pseudo CRISPR spacersfrom an organism—such as a donor organism—into (eg. to modify orreplace) one or more CRISPR spacers, or pseudo CRISPR spacers of a cell.

In one embodiment, one or more CRISPR spacers or pseudo CRISPRspacers—such as one or more CRISPR spacers or pseudo CRISPR spacers froman organism—such as a donor organism—are inserted into (eg. to modify orreplace) one or more CRISPR spacers or pseudo CRISPR spacers of thecell.

In one embodiment, one or more CRISPR spacers or pseudo CRISPRspacers—such as one or more CRISPR spacers or pseudo CRISPR spacers froman organism-such as a donor organism—are inserted into (eg. to modify orreplace) one or more, preferably, two or more CRISPR repeats of thecell. In this embodiment of the invention, it is preferred that at leastone functional CRISPR repeat-cas combination remains intact in the cell.

In one embodiment, one or more CRISPR spacers or pseudo CRISPRspacers—such as one or more CRISPR spacers or pseudo CRISPR spacers froman organism—such as a donor organism—are inserted into (eg. to modify orreplace) the same or different CRISPR spacers of the cell.

In one embodiment, one or more CRISPR spacers or pseudo CRISPRspacers—such as one or more CRISPR spacers or pseudo CRISPR spacers froman organism—such as a donor organism—are inserted adjacent to (eg. tomodify or replace) one or more CRISPR spacers or pseudo CRISPR spacersof the cell.

In the context of the present invention, the term “adjacent” means “nextto” in its broadest sense and includes “directly adjacent”. Thus, in oneembodiment, one or more CRISPR spacers or pseudo CRISPR spacers from anorganism may be inserted “directly adjacent” to one or more CRISPRspacers or pseudo CRISPR spacers of the cell. ie. the CRISPR spacer(s)or pseudo CRISPR spacer(s) is inserted such that there are nointervening nucleotides between the spacers.

In another embodiment, the CRISPR spacer(s) or pseudo CRISPR spacer(s)are inserted such that there are at least 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 10,000, 100,000 or even1,000,000 or more intervening nucleotides between the spacers.

In another embodiment, the intervening nucleotide may be called a leadersequence. These terms are used interchangeably herein. The leadersequence can be of a different length in different bacteria. Suitablythe leader sequence is at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 200, 300, 400 or 500 or more nucleotides inlength. Suitably the leader sequence is between the last cas gene (atthe 3′ end) and the first CRISPR repeat (at the 5′ end) of the CRISPRlocus.

In one embodiment the leader sequence may be between about 20-500nucleotides in length.

In one embodiment, one or more CRISPR spacers or pseudo CRISPRspacers—such as one or more CRISPR spacers or pseudo CRISPR spacers froma donor organism—are inserted adjacent to one or more, preferably, twoor more CRISPR repeats of the cell.

In another embodiment, one or more CRISPR spacers or pseudo CRISPRspacers—such as one or more CRISPR spacers or pseudo CRISPR spacers froma donor organism—are inserted adjacent to one or more cas genes of thecell.

In another embodiment, one or more CRISPR spacers or pseudo CRISPRspacers—such as one or more CRISPR spacers or pseudo CRISPR spacers froma donor organism—are inserted adjacent to the same or different spacersof the recipient cell.

In another embodiment, one or more CRISPR spacers or pseudo CRISPRspacers—such as one or more CRISPR spacers or pseudo CRISPR spacers froma donor organism—are each inserted adjacent to the same or differentCRISPR repeats of the cell.

In another embodiment, one or more CRISPR spacers or pseudo CRISPRspacers—such as one or more CRISPR spacers or pseudo CRISPR spacers froma donor organism—are each inserted adjacent to the same or different casgenes of the recipient cell.

In another embodiment, two or more CRISPR spacers or pseudo CRISPRspacers—such as two or more CRISPR spacers or pseudo CRISPR spacers froma donor organism—are each inserted adjacent to the same or differentCRISPR spacers or pseudo CRISPR spacers and/or CRISPR repeats and/or casgenes of the recipient cell.

In another embodiment, the sequence of the CRISPR spacer—such as one ormore CRISPR spacers from a donor organism—of the recipient cell ismodified such that the CRISPR spacer has homology to the CRISPR spacerof the donor organism.

In another embodiment, the sequence of the spacer of the cell ismodified such that it has homology to the CRISPR spacer or pseudo CRISPRspacer of the organism.

In one embodiment, the CRISPR spacer has 100% homology to the CRISPRspacer of the donor organism.

The CRISPR spacer(s) or pseudo CRISPR spacers may comprise DNA or RNA ofgenomic, synthetic or recombinant origin.

The CRISPR spacer (s) or pseudo CRISPR spacers may be double-stranded orsingle-stranded whether representing the sense or antisense strand orcombinations thereof.

The CRISPR spacer (s) or pseudo CRISPR spacers may be prepared by use ofrecombinant DNA techniques (e.g. recombinant DNA), as described herein.

The modification may comprise inserting one or more CRISPR spacers orpseudo CRISPR spacers from an organism—such as a donor organism—that issubstantially resistant to a target nucleic acid or a transcriptionproduct thereof into one or more CRISPR loci of a substantiallysensitive cell.

The modification may comprise inserting one or more CRISPR spacers orpseudo CRISPR spacers from an organism—such as a donor organism—that issubstantially resistant to a target nucleic acid or a transcriptionproduct thereof into (eg. between) a functional combination of at leasttwo CRISPR repeats and at least one cas gene in a substantiallysensitive cell.

The modification may even comprise modifying (eg. mutating) the DNA of acell (eg. a recipient cell)—such as plasmid DNA or genomic DNA—such thatone or more cas genes are created in the DNA of the cell. By way offurther example, the cas genes may be cloned into a construct, a plasmidor a vector and the like which is then transformed into the cell, usingmethods such as those described herein.

The modification may even comprise modifying (eg. mutating) the DNA of acell (eg. a recipient cell)—such as plasmid DNA or genomic DNA—such thatone or more, preferably, two or more CRISPR repeats are created in theDNA of the cell. By way of further example, the CRISPR repeats may becloned into a construct, a plasmid or a vector and the like which isthen transformed into the cell, using methods such as those describedherein.

The modification may even comprise modifying (eg. mutating) the DNA of acell (eg. a recipient cell)—such as plasmid DNA or genomic DNA—such thatone or more cas-CRISPR repeat functional combinations are created in theDNA of the cell. By way of further example, the cas-CRISPR repeatfunctional combinations may be cloned into a construct, a plasmid or avector and the like which is then transformed into the cell, usingmethods such as those described herein.

The modification may even comprise modifying (eg. mutating) the DNA of acell (eg. a recipient cell)—such as plasmid DNA or genomic DNA—such thatone or more CRISPR spacers are created in the DNA of the cell. By way offurther example, the CRISPR spacers may be cloned into a construct, aplasmid or a vector and the like which is then transformed into thecell, using methods such as those described herein.

In one embodiment, a CRISPR spacer is flanked by two CRISPR repeats. Inother words, a CRISPR spacer has at least one CRISPR repeat on eachside.

Suitably, the modification comprises inserting one or more CRISPRspacers (eg. heterologous CRISPR spacers) in the vicinity of (eg.adjacent to, suitably, directly adjacent to) one or more cas genesand/or the leader sequence. Suitably, according to this embodiment ofthe present invention, the organisation of the naturally occurringCRISPR locus is maintained following insertion of the one or more CRISPRspacers.

Cluster

It has also been surprisingly found that it is not possible to merelyexchange CRISPR repeat-cas combinations between any cells (eg. anystrains, species or genera of cells) since it is believed that this willnot necessarily result in functional CRISPR repeat-cas combinations.

Rather, for the CRISPR repeat-cas combination(s) to be functional theyshould be compatible. Accordingly, it is believed that it is notpossible to switch cas genes or CRISPR repeats between different CRISPRloci unless they are from the same cluster.

Even more surprising is that the clusters do not follow the “organism”phylogeny. Specifically, within one organism, there may be more than oneCRISPR. These CRISPR(s) can belong to different clusters, even thoughthey are present in the same organism. As a result, it is believed thata functional CRISPR repeat-cas. combination requires that thecombination be switched within a cluster as opposed to within anorganism.

For the avoidance of doubt, the term “cluster” as used herein does notrefer to a—cluster of genes located at the same locus (typically formingan operon) but to the output from sequence comparison analysis—such asmultiple sequence comparison analysis and/or multiple sequencealignments and/or dot plot analysis. Accordingly, cluster analysis ofCRISPR loci may be performed using various methods that are known in theart—such as dot-plot analysis as taught herein below for example ormultiple alignment followed by dendrogram calculation.

Advantageously, the use of naturally co-occurring CRISPR repeat-cascombination(s) provides for the interchange of the combination bothwithin and between a given species, thereby making it possible toengineer the resistance of one strain using the combination from adifferent strain.

The cluster may be a class, a family or a group of sequences.

Determining Resistance

In a further aspect, there is provided a method for determining theresistance profile of a cell against a target nucleic acid. As usedherein, the term “resistance profile” means one or more entities againstwhich the cell is sensitive or resistant. Accordingly, the resistanceprofile of a cell may be that the cell is resistant to a firstbacteriophage, sensitive to a second bacteriophage, resistant to a firstmobile genetic element and sensitive to a first antibiotic resistancegene etc.

One or more cas genes or proteins, and/or one or more, preferably, twoor more CRISPR repeats and/or one or more CRISPR spacers etc. within acell may be detected or sequenced so as to predict/determine the likelyresistance profile of a particular cell.

Suitably, one or more CRISPR spacers within a cell are detected orsequenced so as to predict/determine the likely resistance profile of aparticular cell.

Suitable detection methods may include PCR, DNA-DNA hybridization (orDNA-RNA hybridization ie. using DNA or RNA probes that could besynthetic, labelled oligonucleotides, for example). DNA microarrays mayalso be used.

One or more cas -CRISPR repeat functional combinations and/or one ormore CRISPR spacers within a cell may be detected or sequenced so as topredict/determine the likely resistance profile of a particular cell. Byway of example, it is possible to predict/determine the likelyresistance profile of a particular bacterial cell to one or morebacteriophage which can be used as a lysotype predictor for microbialselection.

One or more Cas genes and/or one or more CRISPR repeats may be sequencedin addition to one or more CRISPR spacers in order to verify thecompatibility of the Cas gene-CRISPR repeat combination or even toidentify new pairs of compatible cas/repeats.

Recipient Cell

As used herein, the term “recipient cell” refers to any cell in whichresistance against a target nucleic acid or a transcription productthereof is modulated or is to be modulated.

In one embodiment, the recipient cell refers to any cell comprising therecombinant nucleic acid according to the present invention.

The recipient cell may comprise one or more, preferably, two or moreCRISPR repeats and one or more cas genes or proteins. Suitably, theCRISPR repeats and the cas genes or proteins form a functionalcombination in the recipient cell, as described herein.

The recipient cell may comprise one or more modified CRISPR repeatsand/or one or more modified cas genes or proteins. Suitably, themodified CRISPR repeats and/or the modified cas genes or proteins form afunctional combination in the recipient cell, as described herein.

The recipient cell may comprise one or more genetically engineeredCRISPR repeats and/or one or more genetically engineered cas genes orproteins. Suitably, the genetically engineered CRISPR repeats and/or thegenetically engineered cas genes or proteins form a functionalcombination in the recipient cell, as described herein.

The recipient cell may comprise one or more recombinant CRISPR repeatsand/or one or more recombinant cas genes or proteins. Suitably, therecombinant CRISPR repeats and/or the recombinant cas genes or proteinsform a functional combination in the recipient cell, as describedherein.

The recipient cell may comprise one or more naturally occurring CRISPRrepeats and one or more naturally occurring cas genes or proteins.Suitably, the CRISPR repeats(s) and the cas gene(s) or proteins form afunctional combination.

By “naturally occurring” we mean occurring naturally in nature.

The recipient cell may even comprise combinations of one or moremodified, genetically engineered, recombinant or naturally occurringCRISPR repeats and one or more modified, genetically engineered,recombinant or naturally occurring cas genes or proteins. Suitably, theone or more modified, genetically engineered, recombinant or naturallyoccurring CRISPR spacer(s) or the one or more modified, geneticallyengineered, recombinant or naturally occurring cas gene(s) or proteinsform a functional combination.

Suitably, the recipient cell is a prokaryotic cell.

Suitably, the recipient cell is a bacterial cell. Suitable bacterialcells are described herein.

The bacterial cell may be selected from a lactic acid bacteria species,a Bifidobacterium species, a Brevibacterium species, a Propionibacteriumspecies, a. Lactococcus species, a Streptococcus species, aLactobacillus species including the Enterococcus species, Pediococcusspecies, a Leuconostoc species and Oenococcus species.

Suitable species include, but are not limited to Lactococcus lactis,including Lactococcus lactis subsp. lactis and Lactococcus lactis subsp.cremoris, Lactococcus lactis subsp. cremoris, Leuconostoc sp.,Lactococcus lactis subsp. lactis biovar, Streptococcus thermophilus,Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillushelveticus, Bifidobacterium lactis, Lactobacillus acidophilus,Lactobacillus casei.

The cell in which resistance is to be modulated may be a bacterial cellused for the fermentation of meat (including beef, pork, and poultry)including, but not limited to, lactic acid bacteria, Pediococcuscerevisiae, Lactobacillus plantarum, Lactobacillus brevis, Micrococcusspecies, Lactobacillus sakei, Lactobacillus curvatus, Pediococcuspentosaceus, Staphylococcus xylosus and Staphylococcus vitulinus andmixtures thereof (Food Biotechnology, 538-39 (D. Knorr Ed. 1987); C.Pederson, Microbiology of Fermented Foods, 210-34 (2d ed. 1979); U.S.Pat. No. 2,225,783).

The cell in which resistance is to be modulated may be a bacterial cellused for the fermentation of vegetables (e.g., carrots, cucumbers,tomatoes, peppers, and cabbage) including, but not limited to,Lactobacillus plantatum, Lactobacillus brevis, Leuconostocmesenteroides, Pediococcus pentosaceus, and mixtures thereof (FoodBiotechnology, 540 (D. Knorr Ed. 1987); C. Pederson, Microbiology ofFermented Foods, 153-209 (2d ed. 1979); U.S. Pat. No. 3,024,116; U.S.Pat. No. 3,403,032; U.S. Pat. No. 3,932,674; and U.S. Pat. No.3,897,307).

The cell in which resistance is to be modulated may be a bacterial cellused for the fermentation of dough formed from cereals (e.g., wheat,rye, rice, oats, barley, and corn).

The cell in which resistance is to be modulated may be a bacterial cellused for the production of wine. Typically, this is achieved by thefermentation of fruit juice, typically grape juice.

The cell in which resistance is to be modulated may be a bacterial cellused for the fermentation of milk to produce cheese—such asLactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus,Streptococcus thermophilus, Lactococcus lactis subsp. lactis,Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactisbiovar diacetylactis, Bifidobacteria and Enterococci etc and mixturesthereof (Food Biotechnology, 530 (D. Knorr Ed. 1987); C. Pederson,Microbiology of Fermented Foods, 135-51 (2d ed. 1979)).

The cell in which resistance is to be modulated may be a bacterial cellused for the fermentation of milk to produce cheese—such asLactobacillus bulgaricus, Lactobacillus helveticus, Streptococcusthermophilus, Lactococcus lactis subsp. lactis, Lactococcus lactissubsp. cremoris, Lactococcus lactis subsp. lactis biovar, Lactococci,Bifidobacteria and Enterococci etc and mixtures thereof (FoodBiotechnology, 530 (D. Knorr Ed. 1987); C. Pederson, Microbiology ofFermented Foods, 135-51 (2d ed. 1979)). The cell in which resistance isto be modulated may be a bacterial cell used for the fermentation ofegg—such as Pediococcus pentosaceus, Lactobacillus plantarum, andmixtures thereof (Food Biotechnology, 538-39 (D. Knorr Ed. 1987)).

The cell in which resistance is to be modulated may be a bacterium thatnaturally comprises one or more CRISPR loci. CRISPR loci have beenidentified in more than 40 prokaryotes (Jansen et al. 2002b; Mojica etal., 2005; Haft et al., 2005) including, but not limited to Aeropyrum,Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium,Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus,Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex,Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus,Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus,Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter,Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia,Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium,Salmonella, Xanthamonas, Yersinia, Treponema and Thermotoga.

The cell in which resistance is to be modulated may be a bacterium foruse in cosmetic or pharmaceutical compositions. Such compositions maycomprise a microbial culture and/or labelled bacterium and/or a. cellculture according to the present invention. Thus the microbial cultureand/or labelled bacterium and/or a cell culture according to the presentinvention may be compatible in cosmetics or in pharmacy or in therapy.

Donor Organism

In one embodiment, the term “donor-organism” refers to an organism orcell from which the CRISPR repeat and/or cas gene and/or combination(s)thereof and/or CRISPR spacers are derivable (preferably, derived). Thesecan be the same or different.

In one embodiment, the term “donor organism” refers to an organism orcell from which the one or more, preferably, two or more CRISPR repeatsand/or one or more cas gene and/or combination(s) thereof and/or CRISPRspacers are derivable (preferably, derived). These can be the same ordifferent.

In one embodiment, the CRISPR spacer or pseudo CRISPR spacer issynthetically derived.

In one embodiment, the donor organism or cell comprises one or moreCRISPR spacers, which confers the specific of immunity against a targetnucleic acid or transcription product thereof.

In one embodiment, the donor organism or cell from which the cas geneand/or CRISPR repeat and/or combination thereof is derivable (preferablyderived) is also the recipient cell/organism for the recombinant CRISPRlocus. These can be the same or different.

In one embodiment, the donor organism or cell from which the CRISPRspacer is derivable (preferably derived) is also the recipientcell/organism for the recombinant CRISPR locus. These can be the same ordifferent.

When it is the case that the donor organism is a bacterial cell then thedonor organism will typically comprise a CRISPR spacer which confers thespecific immunity against the target nucleic acid or transcriptionproduct thereof.

The organism may be a bacterial cell or a bacteriophage.

Suitably, the organism is a bacteriophage.

Host Cells

As used herein, the term “host cell” refers to any cell that comprisesthe combination, the construct or the vector and the like according tothe present invention.

Host cells may be transformed or transfected with a nucleotide sequencecontained in a vector e.g. a cloning vector. Said nucleotide sequencemay be carried in a vector for the replication and/or expression of thenucleotide sequence. The cells will be chosen to be compatible with thesaid vector and may, for example, be prokaryotic (for example bacterial)cells.

Aspects of the present invention also relate to host cells comprisingthe combination, construct or the vector of the present invention. Theconstruct or the vector may comprise a nucleotide sequence forreplication and expression of the sequence. The cells will be chosen tobe compatible with the vector and may, for example, be prokaryotic (forexample bacterial) cells.

Construct

In a further aspect, there is provided a construct comprising one ormore of the nucleic acid sequences described herein.

The term “construct”—which is synonymous with terms such as “conjugate”,“cassette” and “hybrid”—includes a nucleotide sequence directly orindirectly attached to another sequence—such as a regulatory sequence(eg. a promoter). By way of example, the present invention covers aconstruct comprising a nucleotide sequence operably linked to such aregulatory sequence. The term “operably linked” refers to ajuxtaposition wherein the components described are in a relationshippermitting them to function in their intended manner. A regulatorysequence “operably linked” to a coding sequence is ligated in such a waythat expression of the coding sequence is achieved under conditioncompatible with the control sequences.

The term “regulatory sequences” includes promoters and enhancers andother expression regulation signals.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site.

The construct may even contain or express a marker, which allows for theselection of the nucleotide sequence construct in, for example, abacterium. Various markers exist which may be used, for example thosemarkers that provide for antibiotic resistance—e.g. resistance tobacterial antibiotics—such as Erythromycin, Ampicillin, Streptomycin andTetracycline.

Vector

The construct may be or may be included in a vector (eg. a plasmid).

Thus, in a further aspect there is provided a vector comprising one ormore of the constructs or sequences described herein.

The term “vector” includes expression vectors and transformation vectorsand shuttle vectors.

The term “transformation vector”, means a construct capable of beingtransferred from one entity to another entity—which may be of thespecies or may be of a different species. If the construct is capable ofbeing transferred from one species to another then the transformationvector is sometimes called a “shuttle vector”.

The vectors may be transformed into a suitable cell (eg. a host cell) asdescribed below.

The vectors may be for example, plasmid or phage vectors provided withan origin of replication, optionally a promoter for the expression ofthe said polynucleotide and optionally a regulator of the promoter.

The vectors may contain one or more selectable marker nucleotidesequences. The most suitable selection systems for industrialmicro-organisms are those formed by the group of selection markers whichdo not require a mutation in the host organism.

The vectors may be used in vitro, for example for the production of RNAor used to transfect or transform a host cell.

Thus, polynucleotides may be incorporated into a recombinant vector(typically a replicable vector), for example, a cloning or expressionvector. The vector may be used to replicate the nucleic acid in acompatible host cell.

Transfection

Introduction of a nucleic acid (eg. a construct or vector) into a cellcan be effected by various methods. For example, calcium phosphatetransfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction or infectionmay be used. Such methods are described in many standard laboratorymanuals—such as Sambrook et al., Molecular Cloning: A Laboratory Manual,2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.

Cells containing the nucleic acid (eg. a construct or vector) may beselected by using, for example, Erythromycin for cells transfected witha nucleic acid (eg. a construct or vector) carrying a resistanceselectable marker.

Transformation

Teachings on the transformation of cells are well documented in the art,for example see Sambrook et al (Molecular Cloning: A Laboratory Manual,2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel etal., Current Protocols in Molecular Biology (1995), John Wiley & Sons,Inc.

A cell may be transformed with a nucleic acid (eg. a construct orvector). Cells transformed with the nucleotide sequence may be culturedunder conditions suitable for the replication or expression of thenucleotide sequence.

Introducing

In the context of introducing a nucleic acid into a cell, in oneembodiment it is preferred that the term “introducing” means one or moreof transforming, transfecting, conjugating or transducing.

Starter Cultures

Starter cultures are used extensively in the food industry in themanufacture of fermented products including milk products—such asyoghurt and cheese, meat products, bakery products, wine and vegetableproducts.

Starter cultures used in the manufacture of many fermented milk, cheeseand butter products include cultures of bacteria, generally classifiedas lactic acid bacteria. Such bacterial starter cultures impart specificfeatures to various dairy products by performing a number of functions.

Commercial non-concentrated cultures of bacteria are referred to inindustry as ‘mother cultures’, and are propagated at the productionsite, for example a dairy, before being added to an edible startingmaterial, such as milk, for fermentation. The starter culture propagatedat the production site for inoculation into an edible starting materialis referred to as the ‘bulk starter’.

Suitable starter cultures for use in the present invention may includeany organism which is of use in the food, cosmetic or pharmaceuticalindustry.

For example, the starter culture may be suitable for use in the dairyindustry. When used in the dairy industry the starter culture may beselected from a lactic acid bacteria species, a Bifidobacterium species,a Brevibacterium species, a Propionibacterium species. Suitable startercultures of the lactic acid bacteria group include commonly used strainsof a Lactococcus species, a Streptococcus species, a Lactobacillusspecies including the Lactobacillus acidophilus, Enterococcus species,Pediococcus species, a Leuconostoc species and Oenococcus species.

Cultures of lactic acid bacteria are commonly used in the manufacture offermented milk products—such as buttermilk, yoghurt or sour cream, andin the manufacture of butter and cheese, for example Brie or Harvati.Lactococcus species include the widely used Lactococcus lactis,including Lactococcus lactis subsp. lactis and Lactococcus lactis subsp.cremoris.

Other lactic acid bacteria species include Leuconostoc sp.,Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricusand Lactobacillus helveticus. In addition, probiotic strains—such asLactococcus species—include the widely used Lactococcus lactis,including Lactococcus lactis subsp. lactis and Lactococcus lactis subsp.cremoris. Mesophilic cultures of lactic acid bacteria commonly used inthe manufacture of fermented milk products such as buttermilk, yoghurtor sour cream, and in the manufacture of butter and cheese, for exampleBrie or Harvati. Other Lactococcus species include Lactococcus lactissubsp. cremoris, Lactococcus lactis, Leuconostoc sp., Lactococcus lactissubsp. lactis biovar, Streptococcus thermophilus, Lactobacillusdelbrueckii subsp. bulgaricus and Lactobacillus helveticus. In addition,probiotic strains such as Bifidobacterium lactis, Lactobacillusacidophilus, Lactobacillus casei may be added during said manufacturingto enhance flavour or to promote health.

Cultures of lactic acid bacteria commonly used in the manufacture ofcheddar and Monterey Jack cheeses include Streptococcus thermophilus,Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremorisor combinations thereof.

Thermophilic cultures of lactic acid bacteria commonly used in themanufacture of Italian cheeses such as Pasta filata or parmesan, includeStreptococcus thermophilus and Lactobacillus delbrueckii subspbulgaricus. Other Lactobacillus species—such as Lactobacillushelveticus—may be added during manufacturing to obtain a desiredflavour.

Advantageously, the starter culture organism may comprise or consist ofa genetically modified strain (prepared according to the methods desiredherein) of one of the above lactic acid bacteria strains or any otherstarter culture strain.

The selection of organisms for the starter culture of the invention willdepend on the particular type of products to be prepared and treated.Thus, for example, for cheese and butter manufacturing, mesophilliccultures of Lactococcus species, Leuconostoc species and Lactobacillusspecies are widely used, whereas for yoghurt and other fermented milkproducts, thermophillic strains of Streptococcus species and ofLactobacillus species are typically used.

The starter culture may even be a dried starter culture.

The starter culture may be a concentrated starter culture.

The starter culture may be a concentrated starter culture used in directinoculation.

The starter culture may be a frozen starter culture.

The starter culture may consist of one bacterial strain, ie., a pureculture. In this case, substantially. all, or at least a significantportion of the bacterial starter culture would generally comprise thesame bacterium.

In the alternative, the starter culture may comprise several bacterialstrains, ie., a defined mixed culture.

Lactic Acid Bacteria

Particularly suitable starter cultures, in particular dried startercultures, for use in the present invention comprise lactic acidbacteria.

As used herein the term “lactic acid bacteria” refers to Gram positive,microaerophillic or anaerobic bacteria which ferment sugar with theproduction of acids including lactic acid as the predominantly producedacid, acetic acid, formic acid and propionic acid. The industrially mostuseful lactic acid bacteria are found among Lactococcus species, such asLactococcus lactis, Lactobacillus species, Bifidobacterium species,Streptococcus species, Leuconostoc species, Pediococcus species andPropionibacterium species.

The starter cultures of the present invention may comprise one or morelactic acid bacteria species such as, Lactococcus lactis, Lactobacillusdelbrueckii subsp. bulgaricus and Streptococcus thermophilus orcombinations thereof.

Lactic acid bacteria starter cultures are commonly used in the foodindustry as mixed strain cultures comprising one or more species. For anumber of mixed strain cultures, such as yoghurt starter culturescomprising strains of Lactobacillus delbrueckii subsp. bulgaricus andStreptococcus thermophilus, a symbiotic relationship exists between thespecies wherein the production of lactic acid is greater compared tocultures of single strain lactic acid bacteria (Rajagopal et al. J.DairySci., 73, p.894-899, 1990).

Preparing Starter Cultures

Starter cultures may be prepared by techniques well known in the artsuch as those disclosed in U.S. Pat. No. 4,621,058. By way of example,starter cultures may be prepared by the introduction of an inoculum, forexample a bacterium, to a growth medium to produce an inoculated mediumand ripening the inoculated medium to produce a starter culture.

Preparing Dried Starter Cultures

Dried starter cultures may be prepared by techniques well known in theart, such as those discussed in U.S. Pat. No. 4,423,079 and U.S. Pat.No. 4,140,800.

Dried starter cultures for use in the present invention may be in theform of solid preparations. Examples of solid preparations include, butare not limited to tablets, pellets, capsules, dusts, granules andpowders which may be wettable, spray-dried, freeze-dried or lyophilised.

The dried starter cultures for use in the present. invention may be ineither a deep frozen pellet form or freeze-dried powder form. Driedstarter cultures in a deep frozen pellet or freeze-dried powder form maybe prepared according to the methods known in the art.

The starter cultures for use in the present invention may be in the formof concentrates which comprise a substantially high concentration of oneor more bacteria. Suitably the concentrates may be diluted with water orresuspended in water or other suitable diluents, for example, anappropriate growth medium or mineral or vegetable oils, for use in thepresent invention. The dried starter cultures of the present inventionin the form of concentrates may be prepared according to the methodsknown in the art, for example by centrifugation, filtration or acombination of such techniques.

Product

Suitable products for use in the present invention include, but are notlimited to, a foodstuffs, cosmetic products or pharmaceutical products.

Any product, which is prepared from, or comprises, a culture iscontemplated in accordance with the present invention. These include,but are not limited to, fruits, legumes, fodder crops and vegetablesincluding derived products, grain and grain-derived products, dairyfoods and dairy food-derived products, meat, poultry, seafood, cosmeticand pharmaceutical products.

The term “food” is used in a broad sense and includes feeds, foodstuffs,food ingredients, food supplements, and functional foods.

As used herein the term “food ingredient” includes a formulation, whichis or can be added to foods and includes formulations which can be usedat low levels in a wide variety of products that require, for example,acidifying or emulsifying.

As used herein, the term “functional food” means a food which is capableof providing not only a nutritional effect and/or a taste satisfaction,but is also capable of delivering a further beneficial effect toconsumer. Although there is no legal definition of a functional food,most of the parties with an interest in this area agree that there arefoods marketed as having specific health effects.

The term “food” covers food for humans as well as food for animals (i.e.a feed). In a preferred aspect, the food is for human consumption.

The cells described herein may be—or may be added to—a food ingredient,a food supplement, or a functional food.

The food may be in the form of a solution or as a solid—depending on theuse and/or the mode of application and/or the mode of administration.

The cells described herein can be used in the preparation of foodproducts such as one or more of: confectionery products, dairy products,meat products, poultry products, fish products and bakery products.

By way of example, the bacterium can be used as ingredients to softdrinks, a fruit juice or a beverage comprising whey protein, healthteas, cocoa drinks, milk drinks and lactic acid bacteria drinks,yoghurt, drinking yoghurt and wine.

There is also provided a method of preparing a food, the methodcomprising admixing the cells according to the present invention with afood ingredient (such as a starting material for a food). The method forpreparing a food is also another aspect of the present invention.

Suitably a food as described herein is a dairy product. More preferablya dairy product as described herein is one or more of the following: ayoghurt, a cheese (such as an acid curd cheese, a hard cheese, asemi-hard cheese, a cottage cheese), a buttermilk, quark, a sour cream,kefir, a fermented whey-based beverage, a koumiss, a milk drink and ayoghurt drink.

Here, the term “food” is used in a broad sense—and covers food forhumans as well as food for animals (i.e. a feed). In a preferred aspect,the food is for human consumption.

The term feed as used herein includes raw and processed plant materialand non plant material. The feed may be any feed suitable forconsumption by an animal, including livestock (animal) feed, for examplepoultry feed, fish feed or crustacean feed for example.

Variants/Homologues/Derivatives/Fragments

The present invention encompasses the use of variants, homologues,derivatives and fragments thereof, including variants, homologues,derivatives and fragments of CRISPR loci, CRISPR spacers, pseudo CRISRspacers, cas genes or proteins, CRISPR repeats, functional CRISPRrepeat-cas gene combinations and target nucleic acid sequences ortranscription products thereof.

The term “variant” is used to mean a naturally occurring polypeptide ornucleotide sequences which differs from a wild-type sequence.

The term “fragment” indicates that, a polypeptide or nucleotide sequencecomprises a fraction of a wild-type sequence. It may comprise one ormore large contiguous sections of sequence or a plurality of smallsections. The sequence may also comprise other elements of sequence, forexample, it may be a fusion protein with another protein. Preferably thesequence comprises at least 50%, more preferably at least 65%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 95%, more preferably at least96%, more preferably at least 97%, more preferably at least 98%, mostpreferably at least 99% of the wild-type sequence.

Preferably, the fragment retains 50%, more preferably 60%, morepreferably 70%, more preferably 80%, more preferably 85%, morepreferably 90%, more preferably 95%, more preferably 96%, morepreferably 97%, more preferably 98%, or most preferably 99% activity ofthe wild-type polypeptide or nucleotide sequence.

Preferably, a CRISPR spacer or pseudo CRISPR spacer comprises at least50%, more preferably at least 65%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 95%, more preferably at least 96%, more preferably at least97%, more preferably at least 98%, most preferably at least 99% of thewild-type sequence. Preferably, a CRISPR spacer retains 50%, morepreferably 60%, more preferably 70%, more preferably 80%, morepreferably 85%, more preferably 90%, more preferably 95%, morepreferably 96%, more preferably 97%, more preferably 98%, or mostpreferably 99% activity of the wild-type polypeptide or nucleotidesequence.

Preferably, a cas gene comprises at least 50%, more preferably at least65%, more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, more preferably at least 95%, more preferablyat least 96%, more preferably at least 97%, more preferably at least98%, most preferably at least 99% of the wild-type sequence. Preferably,a cas gene retains 50%, more preferably 60%, more preferably 70%, morepreferably 80%, more preferably 85%, more preferably 90%, morepreferably 95%, more preferably 96%, more preferably 97%, morepreferably 98%, or most preferably 99% activity of the wild-typepolypeptide or nucleotide sequence.

Preferably, a Cas protein comprises at least 50%, more preferably atleast 65%, more preferably at least 80%, more preferably at least 85%,more preferably at least 90%, more preferably at least 95%, morepreferably at least 96%, more preferably at least 97%, more preferablyat least 98%, most preferably at least 99% of the wild-type sequence.Preferably, a Cas protein retains 50%, more preferably 60%, morepreferably 70%, more preferably 80%, more preferably 85%, morepreferably 90%, more preferably 95%, more preferably 96%, morepreferably 97%, more. preferably 98%, or most preferably 99% activity ofthe wild-type polypeptide or nucleotide sequence.

Preferably, a CRISPR repeat comprises at least 50%, more preferably atleast 65%, more preferably at least 80%, more preferably at least 85%,more preferably at least 90%, more preferably at least 95%, morepreferably at least 96%, more preferably at least 97%, more preferablyat least 98%, most preferably at least 99% of the wild-type sequence.Preferably, a CRISPR repeat retains 50%, more preferably 60%, morepreferably 70%, more preferably 80%, more preferably 85%, morepreferably 90%, more preferably 95%, more preferably 96%, morepreferably 97%, more preferably 98%, or most preferably 99% activity ofthe wild-type polypeptide or nucleotide sequence.

Preferably, a functional CRISPR repeat-cas combination comprises atleast 50%, more preferably at least 65%, more preferably at least 80%,more preferably at least 85%, more preferably at least 90%, morepreferably at least 95%, more preferably at least 96%, more preferablyat least 97%, more preferably at least 98%, most preferably at least 99%of the wild-type sequence. Preferably, functional CRISPR repeat-cascombination retains 50%, more preferably 60%, more preferably 70%, morepreferably 80%, more preferably 85%, more preferably 90%, morepreferably 95%, more preferably 96%, more preferably 97%, morepreferably 98%, or most preferably 99% activity of the wild-typepolypeptide or nucleotide sequence.

Preferably, a target nucleic acid sequence comprises at least 50%, morepreferably at least 65%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least95%, more preferably at least 96%, more preferably at least 97%, morepreferably at least 98%, most preferably at least 99% of the wild-typesequence. Preferably, a target nucleic acid sequence retains 50%, morepreferably 60%, more preferably 70%, more preferably 80%, morepreferably 85%, more preferably 90%, more preferably 95%, morepreferably 96%, more preferably 97%, more preferably 98%, or mostpreferably 99% activity of the wild-type polypeptide or nucleotidesequence.

The fragment may be a functional fragment.

By a “functional fragment” of a molecule is understood a fragmentretaining or possessing substantially the same biological activity asthe intact molecule. In all instances, a functional fragment of amolecule retains at least 10% and at least about 25%, 50%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% of the biological activity of theintact molecule.

The term “homologue” means an entity having a certain homology with thesubject amino acid sequences and the subject nucleotide sequences. Here,the term “homology” can be equated with “identity”.

In the present context, a homologous sequence is taken to include anamino acid sequence, which may be at least 75, 85 or 90% identical,preferably at least 95%, 96%, 97%, 98% or 99% identical to the subjectsequence. Although homology can also be considered in terms ofsimilarity (i.e. amino acid residues having similar chemicalproperties/functions), in the context of the present invention it ispreferred to express homology in terms of sequence identity.

In the present context, a homologous sequence is taken to include anucleotide sequence, which may be at least 75, 85 or 90% identical,preferably at least 95%, 96%, 97%, 98% or 99% identical to the subjectsequence. Although homology can also be considered in terms ofsimilarity (i.e. amino acid residues having similar chemicalproperties/functions), in the context of the present invention it ispreferred to express homology in terms of sequence identity.

Homology comparisons may be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a, sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example, when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410), the GENEWORKSsuite of comparison tools and CLUSTAL. Both BLAST and FASTA areavailable for offline and online searching (see Ausubel et al., 1999ibid, pages 7-58 to 7-60). However, for some applications, it ispreferred to use the GCG Bestfit program. A new tool, called BLAST 2Sequences is also available for comparing protein and nucleotidesequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS MicrobiolLett 1999 177(1): 187-8).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). For some applications, it ispreferred to use the public default values for the GCG package, or inthe case of other software, the default matrix—such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

Should Gap Penalties be used when determining sequence identity, thensuitably the following parameters are used:

FOR BLAST GAP OPEN 0 GAP EXTENSION 0

FOR CLUSTAL DNA PROTEIN WORD SIZE 2 1 K triple GAP PENALTY 10 10 GAPEXTENSION 0.1 0.1

For polypeptide sequence comparison the following settings may be used:GAP creation penalty of 3.0 and GAP extension penalty of 0.1. Suitably,the degree of identity with regard to an amino acid sequence isdetermined over at least 5 contiguous amino acids, determined over atleast 10 contiguous amino acids, over at least 15 contiguous aminoacids, over at least 20 contiguous amino acids, over at least 30contiguous amino acids, over at least 40 contiguous amino acids, over atleast 50 contiguous amino acids, or over at least 60 contiguous aminoacids.

The sequences may also have deletions, insertions or substitutions ofamino acid residues, which produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the secondary binding activity of the substance isretained. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine,glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine, and tyrosine.

Conservative substitutions may be made, for example, according to theTable below. Amino acids in the same block in the second column andsuitably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R AROMATIC H F W Y

The present invention also encompasses homologous substitution(substitution and replacement are both used herein to mean theinterchange of an existing amino acid residue, with an alternativeresidue) may occur i.e. like-for-like substitution—such as basic forbasic, acidic for acidic, polar for polar etc. Non-homologoussubstitution may also occur i.e. from one class of residue to another oralternatively involving the inclusion of unnatural amino acids—such asornithine (hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as O), pyriylalanine, thienylalanine, naphthylalanine andphenylglycine.

Replacements may also be made by unnatural amino acids include; alpha*and alpha-disubstituted* amino acids, N-alkyl amino acids*, lacticacid*, halide derivatives of natural amino acids—such astrifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*,p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyricacid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-aminocaproic acid^(#), 7-amino heptanoic acid*, L-methionine sulfone#*,L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*,L-hydroxyproline^(#), L-thioproline*, methyl derivatives ofphenylalanine (Phe)—such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe(4-amino)^(#), L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionicacid ^(#) and L-Phe (4-benzyl)*. The notation * has been utilised forthe purpose of the discussion above (relating to homologous ornon=homologous substitution), to indicate the hydrophobic nature of thederivative whereas # has been utilised to indicate the hydrophilicnature of the derivative, #* indicates amphipathic characteristics.

Variant amino acid sequences may include suitable spacer groups that maybe inserted between any two amino acid residues of the sequenceincluding alkyl groups—such as methyl, ethyl or propyl groups—inaddition to amino acid spacers—such as glycine or β-alanine residues. Afurther form of variation involves the presence of one or more aminoacid residues in peptoid form will be well understood by those skilledin the art. For the avoidance of doubt, “the peptoid form” is used torefer to variant. amino acid residues wherein the α-carbon substituentgroup is on the residue's nitrogen atom rather than the α-carbon.Processes for preparing peptides in the peptoid form are known in theart, for example, Simon R J et al., PNAS (1992) 89(20), 9367-9371 andHorwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

The nucleotide sequences for use in the present invention may includewithin them synthetic or modified nucleotides. A number of differenttypes of modification to oligonucleotides are known in the art. Theseinclude methylphosphonate and phosphorothioate backbones and/or theaddition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present invention, it is to beunderstood that the nucleotide sequences may be modified by any methodavailable in the art. Such modifications may be carried out to enhancethe in vive activity or life span of nucleotide sequences useful in thepresent invention.

General Recombinant DNA Methodology Techniques

The present invention employs, unless otherwise indicated, conventionaltechniques of chemistry, molecular biology, microbiology, recombinantDNA and immunology, which are within the capabilities of a person ofordinary skill in the art. Such techniques are explained in theliterature. See, for example, J. Sambrook, E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition,Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al.(1995 and periodic supplements; Current Protocols in Molecular Biology;ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J.Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: EssentialTechniques, John Wiley & Sons; M. J. Gait (Editor), 1984,Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M.J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA StructurePart A: Synthesis and Physical Analysis of DNA Methods in Enzymology,Academic Press. Each of these general texts is herein incorporated byreference.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

EXAMPLES Example 1

Insertion of a phage specific spacer into an existing, functional CRISPRto provide resistance to the corresponding phage.

Strain—Streptococcus thermophilus ST0089

Phage—2972

Streptococcus thermophilus ST0089 is an industrially important strainused in the manufacture of yogurt, is genetically amenable tomanipulation, and susceptible to virulent phage 2972. The full genomesequence for phage 2972 was recently determined.

The CRISPR loci is determined in strain ST0089. This is determinedpreferentially by sequencing the entire genome of ST0089. Alternatively,the CRISPR loci is identified via PCR using primer sets with sequencesidentical to S. thermophilus CRISPR elements previously identified.

Once identified, the CRISPR loci sequence is determined as well as theproximal regions which should contain the relevant cas genes.

At least one particular CRISPR-cas locus is selected for furthermanipulation. Functionality of this locus is ascertained through insilico analysis of the spacer regions and their homologies to phage DNAsequences (i.e. absence and/or presence of spacer sequences andcorrelation to phage infectivity with strain ST0089). In the absence ofthis correlation, functionality is assumed based on the presence of alldocumented elements (i.e. repeats, spacers, leader sequences, casgenes—putatively encoding full length proteins).

A suitable spacer sequence(s) is chosen from the genome of phage 2972.The criteria of the selected spacer is based on: 1) length of thespacers within the selected CRISPR locus; 2) about 100% identity to thephage sequence; 3) theoretically any phage sequence may be selected.

In the simplest example, a CRISPR unit consisting of a phage 2972 spacersequence, flanked by two repeating elements (identical to the selectedCRISPR locus) is chemically synthesized. By definition this synthetic“CRISPR unit” is approximately 100 bp in length and is too short forensuing integration into the CRISPR locus.

Therefore, additional flanking DNA is constructed along with the CRISPRunit A minimum of 500 bp of homologous DNA, identical to the targetedCRISPR locus flanks the synthetic CRISPR unit, to facilitateintegration.

There are at least two approaches. One construct emulates the additionof a new spacer onto the existing CRISPR. Alternatively, the entireCRISPR locus is replaced with the synthetic CRISPR unit.

The resulting CRISPR integrant is verified through DNA sequencing of theCRISPR locus prior to biological testing.

Phage sensitivity patterns of the CRISPR integrant against phage 2972 istested and compared with the parental strain.

The constructed CRISPR integrant successfully demonstrates the directcorrelation between the presence of a specific spacer within the propercontext of CRISPR-cas.

Example 2

A spacer homologous to a phage DNA is inserted into a cell—such asrecipient cell. The cell becomes resistant to the phage. In a CRISPRlocus within the selected strain, a new CRISPR spacer is designed fromphage DNA (with 100% identity to phage DNA) within the anti-receptorgene and inserted into the cell. The anti-receptor gene is targetedbecause CRISPR spacers from other strains have been found to showsimilarity to phage anti-receptor genes. Four strains bearing spacersshowing identity to phage anti-receptor genes are resistant to theparticular phage. The mutant is exposed to phage and it becomesresistant to it.

Example 3

A plasmid comprising a CRISPR spacer is prepared, and we show that thisplasmid cannot be transferred into a cell that contains the same spacer,whereas the plasmid without the spacer can be transformed into the cell.

Example 4

A spacer is inserted into an original host, but not in a CRISPR locus,and the resulting mutant retains its sensitivity to the phage, showingthat the spacer needs to be in a particular environment within a CRISPRand cas genes

Example 5

A whole CRISPR repeat-cas combination is inserted into a cell—such as arecipient cell—to provide immunity against incoming nucleic acid.

Example 6

For a particular CRISPR repeat-cas combination present in two differentstrains, the “exchange” of spacers modifies their phenotypes (phagesensitivity/resistance).

Example 7

One or more cas genes (from a functional CRISPR-cas unit) are deleted.Cas genes are necessary for immunity to be provided. Cas mutants arestill sensitive to the phage, despite the presence of the spaceridentical to phage DNA.

Example 8

The deleted cas genes are cloned on a plasmid. It is possible to providethe cas genes in trans to the host. Where the cas gene is knocked out,immunity can be restored.

Example 9

Different cas-CRISPR-repeat combinations are prepared. Not only are casgenes or proteins required, but also, specific cas-CRISPR repeat pairsare required for functionality. When cas genes or proteins are providedfrom another CRISPR locus, the strain remains sensitive to the phage.

Example 10

When a particular CRISPR spacer is deleted from a naturally occurringCRISPR locus, this removes immunity against a given phage and the hostbecomes sensitive (looses resistance) to the phage to which the spaceris homologous to.

Example 11

Integration of a CRISPR spacer into the CRISPR locus of a bacteriumprovides resistance against a bacteriophage that the CRISPR spacer showsidentity to

(A) Streptococcus thermophilus strain DGCC7710RH1Streptococcus thermophilus

Streptococcus thermophilus strain DGCC7710 (deposited at the French“Collection Nationale de Cultures de Microorganismes” under number CNCMI-2423) possesses at least 3 CRISPR loci: CRISPR1, CRISPR2, and CRISPR3.In strains CNRZI066 and LMG18311 for which the complete genome sequenceis known (Bolotin et al., 2004), CRISPR1 is located at the samechromosomal locus: between str0660 (or stu0660) and str0661 (orstu0661).

In strain DGCC7710, CRISPR1 is also located at the same chromosomallocus, between highly similar genes. CRISPR1 of strain DGCC7710 contains33 repeats (including the terminal repeat), and thus 32 spacers.

All these spacers are different from each other. Most of these spacersare new (not yet described within CRISPR loci), but four spacers closeto the CRISPR1 trailer are identical to already known CRISPR1 spacers:

-   -   the 28^(th) spacer of DGCC7710 is 100% identical to the 31^(st)        CRISPR1 spacer of strain CNRZ1575 (Genbank accession number        DQ072991);    -   the 30^(th) spacer of DGCC7710 is 100% identical to the 27^(th)        CRISPR1 spacer of strain CNRZ703 (Genbank accession number        DQ072990);    -   the 31^(st) spacer of DGCC7710 is 100% identical to the 28^(th)        CRISPR1 spacer of strain CNRZ703 (Genbank accession number        DQ072990);    -   the 32^(nd) spacer of DGCC7710 is 100% identical to the 30^(th)        CRISPR1 spacer of strain CNRZ703 (Genbank accession number        DQ072990).

Virulent Bacteriophage

D858 is a bacteriophage belonging to the Siphoviridae family of viruses.Its genome sequence has been completely determined but is not publishedyet. This phage is virulent to S. thermophilus strain DGCC7710.

Phage Resistant Mutant

Streptococcus thermophilus strain DGCC7710RH1 has been isolated as anatural phage resistant mutant using DGCC7710 as the parental strain,and phage D858 as the virulent phage.

CRISPR1 of strain DGCC7710-RH1 contains 34 repeats (including theterminal repeat), and thus 33 spacers. When compared to the CRISPR1sequence of Streptococcus thermophilus strain DGCC7710, the CRISPR1sequence of Streptococcus thermophilus strain DGCC7710-RH1 possesses oneadditional new spacer (and of course one additional repeat which flanksthe new spacer) at one end of the CRISPR locus (ie. close to the leader,at the 5′ end of the CRISPR locus).

All the other spacers of CRISPR1 locus are unchanged.

The CRISPR1 sequence (5′-3′) of strain DGCC7710-RH1 is:

>CRISPR1_DGCC7710-RH1,caaggacagttattgattttataatcactatgtgggtataaaaacgtcaaaatttcatttgagGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACtcaacaattgcaacatcttataacccacttGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACtgtttgacagcaaatcaagattcgaattgtGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaatgacgaggagctattggcacaacttacaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACcgatttgacaatctgctgaccactgttatcGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACacacttggcaggcttattactcaacagcgaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACctgttccttgttcttttgttgtatcttttcGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACttcattcttccgtttttgtttgcgaatcctGTTPTTGTACTCTCAAGATTTAAGTAACTGTACAACgctggcgaggaaacgaacaaggcctcaacaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACCatagagtggaaaactagaaacagattcaaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACataatgccgttgaattacacggcaaggtcaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACgagcgagctcgaaataatcttaattacaagGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACgttcgctagcgtcatgtggtaacgtatttaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACggcgtcccaatcctgattaatacttactcgGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaacacagcaagacaagaggatgatgctatgGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACcgacacaagaacgtatgcaagagttcaagGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACacaattcttcatccggtaactgctcaagtgGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaattaagggcatagaaagggagacaacatgGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACcgatatttaaaatcattttcataacttcatGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACgcagtatcagcaagcaagctgttagttactGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACataaactatgaaattttataatttttaagaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaataatttatggtatagcttaatatcattgGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACtgcatcgagcacgttcgagtttaccgtttcGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACtctatatcgaggtcaactaacaattatgctGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaatcgttcaaattctgttttaggtacatttGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaatcaatacgacaagagttaaaatggtcttGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACgcttagctgtccaatccacgaacgtggatgGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACcaaccaacggtaacagctactttttacagtGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACataactgaaggataggagcttgtaaagtctGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACtaatgctacatctcaaaggatgatcccagaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaagtagttgatgacctctacaatggtttatGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACacctagaagcatttgagcgtatattgattgGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaattttgccccttctttgccccttgactagGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaccattagcaatcatttgtgcccattgagtGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAGTttgattcaacataaaaagccagttcaattgaacttggcttt Legend Leader sequence:5′-caaggacagttattgattttataatcactatgtgggtataaaaacgtcaaaatttcatttgag-3′Integrated sequence comprising a CRISPR Repeat in upper case and a CRISPR spacer (ie. tagging sequence) in lower case. CRISPR Repeats Terminal repeat:  5′ gtttttgtactctcaagatttaagtaactgtacagt-3′Trailer sequence:  5′ ttgattcaacataaaaagccagttcaattgaacttggcttt3′

The sequence of the new spacer exists within the D858 phage genome andis represented herein as SEQ ID No. 534.

The sequence of the spacer is found between positions 31921 and 31950 bp(ie. on the plus strand) of D858's genome (and has 100% identity to theD858 genomic sequence over 30 nucleotides):

spacer     1 tcaacaattgcaacatcttataacccactt    30      |||||||||||||||||||||||||||||| D85831921 tcaacaattgcaacatcttataacccactt 31950

The new spacer that is integrated into the CRISPR1 locus ofStreptococcus thermophilus strain DGCC7710-RH1 confers to this strainresistance to phage D858, as represented in FIG. 5 and Table 1.

(B) Streptococcus thermophilus Strain DGCC7710RH2

Streptococcus thermophilus strain DGCC7710-RH2 has been isolated as anatural phage resistant mutant using Streptococcus thermophilus strainDGCC7710 as the parental strain, and phage D858 as the virulent phage.

CRISPR1 of Streptococcus thermophilus strain DGCC7710-RH2 contains 34repeats (including the terminal repeat), and thus 33 spacers. Whencompared to the CRISPR1 sequence of Streptococcus thermophilus strainDGCC7710, the CRISPR1 sequence of Streptococcus thermophilus strainDGCC7710-RH2 possesses one additional new spacer (and of course oneadditional repeat which flanks the new spacer) at one end of the CRISPRlocus (ie. close to the leader, at the 5′ end of the CRISPR locus). Allthe other spacers of CRISPR1 locus are unchanged.

The CRISPR1 sequence (5′-3′) of strain DGCC7710-RH2 is:

<CRISPR1__DGCC7710-RH2caaggacagttattgattttataatcactatgtgggtataaaaacgtcaaaatttcatttgagGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACttacgtttgaaaagaatatcaaatcaatgaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACtgtttgacagcaaatcaagattcgaattgtGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaatgacgaggagctattggcacaacttacaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACcgatttgacaatctgctgaccactgttatcGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACacacttggcaggcttattactcaacagcgaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACctgttccttgttcttttgttgtatcttttcGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACttcattcttccgtttttgtttgcgaatcctGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACgctggcgaggaaacgaacaaggcctcaacaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACcatagagtggaaaactagaaacagattcaaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACataatgccgttgaattacacggcaaggtcaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACgagcgagctcgaaataatcttaattacaagGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACgttcgctagcgtcatgtggtaacgtatttaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACggcgtcccaatcctgattaatacttactcgGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaacacagcaagacaagaggatgatgctatgGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACcgacacaagaacgtatgcaagagttcaagGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACacaattcttcatccggtaactgctcaagtgGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaattaagggcatagaaagggagacaacatgGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACcgatatttaaaatcattttcataacttcatGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACgcagtatcagcaagcaagctgttagttactGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACataaactatgaaattttataatttttaaga GTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaataatttatggtatagcttaatatcattgGTTITTGTACTCTCAAGATTTAAGTAACTGTACAACtgcatcgagcacgttcgagtttaccgtttcGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACtctatatcgaggtcaactaacaattatgctGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaatcgttcaaattctgttttaggtacatttGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaatcaatacgacaagagttaaaatggtcttGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACgcttagctgtccaatccacgaacgtggatgGTTTTTGTACTCTCAAGATTVAAGTAACTGTACAACcaaccaacggtaacagctactttttacagtGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACataactgaaggataggagcttgtaaagtctGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACtaatgctacatctcaaaggatgatcccagaGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaagtagttgatgacctctacaatggtttatGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACacctagaagcatttgagcgtatattgattgGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaattttgccccttctttgccccttgactagGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACaccattagcaatcatttgtgcccattgagtGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAGTttgattcaacataaaaagccagttcaattgaacttggcttt Legend Leader sequence: 5′caaggacagttattgattttataatcactatgtgggtataaaaacgtcaaaatttcatttgag 3′Integerated sequence comprising a CRISPR Repeat in upper case and aCRISPR spacer (ie. tagging sequence) in lower case. CRISPR RepeatsTerminal repeat:  5′ gtttttgtactctcaagatttaagtaactgtacagt 3′Trailer sequence: 5′ ttgattcaacataaaaagccagttcaattgaacttggcttt3′

It has been shown that the sequence of the new spacer exists within theD858 phage genome.

The sequence of the spacer (represented herein as SEQ ID No. 535) isfound between positions 17215 and 17244 bp (ie. on the plus strand) ofD858's genome (and has 100% identity to the D858 genomic sequence over30 nucleotides):

spacer     1 ttacgtttgaaaagaatatcaaatcaatga    30      |||||||||||||||||||||||||||||| D85817215 ttacgtttgaaaagaatatcaaatcaatga 17244

The new spacer that is integrated into the CRISPR1 locus ofStreptococcus thermophilus strain DGCC7710-RH2 confers to Streptococcusthermophilus strain DGCC7710-RH2 a resistance to phage D858, asrepresented in FIG. 6 and Table 1.

Example 12 Construct Integration and Knockout Materials and MethodsStrains and Plasmids

Streptococcus thermophilus DGCC7710 parent strain, sensitive to phages858 and 2972Streptococcus thermophilus DGCC7778 CRISPR mutant resistant to 858Streptococcus thermophilus DGCC7778cas1KOStreptococcus thermophilus DGCC7778cas4KOStreptococcus thermophilus DGCC7778RTStreptococcus thermophilus DGCC7778RT′Streptococcus thermophilus DGCC7710R2 CRISPR mutant resistant to 2972Streptococcus thermophilus DGCC7710R2S1S2Escherichia coli EC1,000 provides pORI28 (Russell and Klaenhammer, 2001)Escherichia coli pCR2.1TOPO provides pTOPO (Invitrogen catalog#K4500-01)pTOPO is a plasmid used for sub-cloning of the various constructspTOPOcas1ko contains an integral fragment of cas1pTOPOcas4ko contains an integral fragment of cas4pTOPOS1S2 contains the S1S2 spacer constructpTOPO RT contains the RT terminal repeat constructpORI28 is a plasmid used for integration of the various constructs inthe chromosome of Streptococcus thermophilus strains.pORIcas1ko contains an integral fragment of cas1pORIcas4ko contains an integral fragment of cas4pORIS1S2 contains the S1S2 spacer constructpurist contains the RT terminal repeat construct

Primers

Cas1 5′-caaatggatagagaaacgc-3′ and  5′-ctgataaggtgttcgttgtcc-3′ Cas45′-ggagcagatggaatacaagaaagg-3′ and  5′-gagagactaggttgtctcagca-3′S1S2 and RT P1  5′-acaaacaacagagaagtatctcattg-3′ P2 5′-aacgagtacactcactatttgtacg-3′ P35′-tccactcacgtacaaatagtgagtgtactcgtttttgtattctcaagatttaagtaactgtacagtttgattcaacataaaaag-3′ P45′-attecttcatcctcgctttggtt-3′

Strains and phages were obtained from the Danisco Culture Collection, orfrom referenced material (Russell and Klaenhammer, Applied andEnvironmental Microbiology 2001, 67:43691-4364; Levesque et al., Appliedand Environmental Microbiology 2005 71:4057-4068).

Phage preparation, purification and tests were carried out using methodsdescribed previously (Duplessis et al., Virology 2005, 340:192-208;Levesque et al., Applied and Environmental Microbiology 200571:4057-4068).

Streptococcus thermophilus strains were grown at 37C or 42C in M17(Difco Laboratories) supplemented with 0.5% lactose or sucrose. Forphage infection, 10 mM CaCl2 were added to the medium prior to phageinfection, as described previously (Duplessis et al., Virology 2005,340:192-208; Levesque et al., Applied and Environmental Microbiology2005 71:4057-4068).

Enzymes used to carry out restriction digests and PCR were purchasedfrom Invitrogen and used according to the manufacturer's instructions.PCRs were carried out on an Eppendorf Mastercycler Gradient thermocycleras described previously (Barrangou et al., 2002 Applied andEnvironmental Microbiology 68:2877-2884).

Gene inactivation and site-specific plasmid insertion via homologousrecombination in the Streptococcus thermophilus chromosome were carriedout by sub-cloning into the Invitrogen pCR2.1TOPO system, subsequentcloning in the pORI system using Escherichia coli as a host and theconstructs were ultimately purified and transformed into Streptococcusthermophilus as previously described (Russell and Klaenhammer, Appliedand Environmental Microbiology 2001, 67:43691-4364)

(1) RT Construct Integration

Using the RT Construct engineered as shown in FIG. 17, the construct wasinserted just after cas4, as shown in FIG. 18.

The parent DGCC7778 is resistant to phage 858.

The parent has two spacers (S1 and D2) which are identical to phage 858DNA.

The resulting strain (RT) loses resistance to phage 858, as shown inTable 1. This demonstrates that cas genes need to be in the immediatevicinity of the spacer(s) to confer resistance.

(2) Cas1 Knockout

As shown in FIG. 12 the parent DGCC7778 is engineered such that the cas1gene is disrupted. As shown in Table 1, this results in a loss ofresistance, meaning that cas1 is needed to confer resistance.

(3) Cas4 Knockout

As shown in FIG. 12 the parent DGCC7778 is engineered such that the cas4gene is disrupted.

(4) S1S2 Construct Integration

As shown in FIGS. 14-16 the a S1S2 construct is integrated into theparent DGCC7710.

Summary

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats)(a.k.a. SPIDR—SPacer Interspersed Direct Repeats) constitute a family ofrecently described DNA loci widespread throughout prokaryotic genomes.They are constituted of short and highly conserved DNA palindromicrepeats which are regularly interspaced by highly polymorphic sequencesof about the same length. Additionally, cas genes (CRISPR-associatedgenes) are usually present in the vicinity of CRISPR sequences. In theliterature no clear physiological function has been attributed yet toCRISPR sequences or cas genes.

Here we suggest that CRISPR sequences in combination with cas genes maybe used to provide resistance against incoming nucleic acid.Particularly, we propose that the spacers within CRISPR loci provide thespecificity for immunity against incoming nucleic acid. As a result, wesuggest that cas genes in association with CRISPR sequences be used toprovide cells with resistance against particular nucleic acidsequences—such as bacteriophages, plasmids, transposons, and insertionsequences. Additionally, these elements can be manipulated to generatetargeted immunity against particular nucleic acid sequences, such asphage components, antibiotic resistance genes, virulence factors, novelsequences, undesirable elements and the like. Thus, the simple knowledgeof inter alia CRISPR spacer sequences for a given bacterial strain wouldbe an advantage to determine its lysotype (the lysotype defines theresistance/sensitivity of a given bacterium to various bacteriophages)and predict its ability to survive exposure to defined nucleic acidsequences. Consequently, characterisation of CRISPR loci in bacteriacould help to determine, predict and modify host-phage interaction.Particular application of CRISPR genetic engineering, by addition,deletion or modification of spacer sequences, could lead to phageresistant bacterial variants.

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats),also known as SPIDR (SPacer Interspersed Direct Repeats), form a newfamily of repeated sequences which have been identified in completegenome sequences, in numerous prokaryotes, mostly on chromosomes butalso on plasmids (Mojica et al., 2000; Jansen et al., 2002a). CRISPRloci are constituted of short and highly conserved DNA repeats (24 to 40bp, repeated from 1 to 140 times) which are partially palindromic. Whilethere are certain limits to the base degeneracy between repeats fromdifferent loci and species, there is no absolute conserved sequencethroughout all observed repeats. Moreover the repeats are seeminglyoriented within a particular locus, with regards to the neighbouringgenes. These repeated sequences (usually specific to a species) areinterspaced by polymorphic sequences of constant length (20 to 58 bpdepending on the CRISPR) which are designated as “spacers”. Up to 20different CRISPR loci have been found within a single chromosome. FIG. 1describes one of the CRISPR identified in Streptococcus thermophilusCNRZ1066.

For example, the genome of S. thermophilus LMG18311 contains 3 CRISPRloci. The 36-bp repeated sequences are different in CRISPR1 (34repeats), CRISPR2 (5 repeats), and CRISPR3 (one single sequence);nevertheless, they are perfectly conserved within each locus. CRISPR1and CRISPR2 repeats are respectively interspaced by 33 and 4 sequencesof 30 bp in length. All these spacers are different from each other(apart from minor exceptions: very few spacers may be present twicewithin a given CRISPR locus). They are also different from those foundin other strains—such as CNRZ1066 (41 spacers within CRISPR1) or LMD-9(16 spacers within CRISPR1 and 8 within CRISPR3), which are S.thermophilus strains that have very similar genomes.

Although the biological function of CRISPR loci is unknown somehypotheses have been proposed. For example, it has been proposed thatthey may be involved in the attachment of the chromosome to a cellularstructure, or in the chromosome replication and replicon. partitioning,but no experimental demonstration has been reported to confirm thesehypotheses.

Generally CRISPR loci. are immediately adjacent to a group of 4 to 7.genes which have been called cas (CRISPR-associated) genes (Jansen etal., 2002b). At the present time no clear physiological role has beenattributed to Cas proteins, but for some of them the presence ofparticular protein motifs suggests that they could act as a DNA gyraseor a DNA polymerase. These clusters of 4 to 7 cas genes, eitheroriginating from different loci within a given genome or originatingfrom different microorganisms, can be distinguished and grouped intodifferent types on the basis of sequence similarity. One of our majorfindings is that a given set of cas genes is always associated with agiven repeated sequence within a particular CRISPR locus. In otherwords, cas genes [or Cas proteins] seem to be specific for a. given DNArepeat, meaning that cas genes [or Cas proteins] and the repeatedsequence could form a functional pair. Dotplot analyses indicate thatthe clusters and groups obtained when analyzing Cas protein sequencesare similar to those obtained when analyzing CRISPR repeats (as shown inFIG. 2).

In S. thermophilus, a bacterial species for which several phage genomeshave been sequenced, the 30-bp spacers are often identical to phage DNA(FIG. 3). This observation has also been made for the spacer sequencesof many other bacterial genera and species for which phage DNA sequencesare known. Furthermore it has been previously mentioned in at least tworecent publications (Pourcel et al., 2005; Mojica et al., 2005). On theother hand the absence of significant sequence similarity for theremaining spacer sequences may be explained by the fact that only a fewphage genomes are available at this time. On the basis of very high DNAsequence similarities between some CRISPR spacers and bacteriophagesequences, we propose that the specificity of the CRISPR spacersparticipates in the determination of the strain lysotype. To support theproposal of an effect of CRISPR spacer sequences on the bacterialimmunity against bacteriophages, it was found that a significantproportion of matches for spacers in bacteriophage genome sequencesoccur within genes likely involved in the host specificity (see FIG. 3).Another hypothesis could be that the spacer sequences are recognized bythe bacterium as foreign DNA. Thus, the bacterium would eliminate thenucleic acid molecule bearing this sequence when entering the cell. Oneargument supporting this idea is the deduced peculiar structure ofCRISPR. Indeed, we propose that the repeat elements provide a structuralfeature while the spacers containing the sequence providing specificimmunity against incoming nucleic acid. The palindromic repeats have thepotential to form very stable hairpin (stem-loop) structures (see FIG.4), and they are separated by spacers whose size corresponds to roughly3 turns of the DNA helix (although it can vary between 2 and 5). Thusany CRISPR locus could be highly structured into a series of regularlyspaced DNA hairpins.

Advantageously, the lysotype of a given bacterial strain may be modifiedeither by natural generation of resistant derivatives (BacteriophageInsensitive Mutants), or by genetic engineering. Specifically, geneticengineering solutions may be designed by, for example, addition, bydeletion, or by modification of the spacer sequences or even a completeCRISPR locus.

Examples of applications of this invention include, but are not limitedto:

(i) Phage resistance. Particular CRISPR spacers derived frombacteriophage DNA may be added within a bacterial host CRISPR locus asto provide resistance against this particular bacteriophage, thuspreventing phage attack. Additionally, particular regions within thephage genome (host specificity proteins) can be targeted that provideparticular phage-host recognition, or that are highly conserved withinphage DNA, such as sequences from helicase or primase genes, head andtail structural proteins, or proteins with conserved domains (eg.helicase, holing, lysine, and others) or conserved sequences amongstimportant phage genes.(ii) Resistance to plasmid transfer. Particular CRISPR spacers derivedfrom plasmid DNA can be added within a bacterium CRISPR locus as toprovide resistance against this particular plasmid, thus preventingtransfer of foreign DNA into the microbe. Specifically, particularregions within the plasmid DNA can be targeted as to provide immunityagainst plasmid DNA, such as sequences within the plasmid's origin ofreplication.(iii) Resistance to mobile genetic elements. Particular CRISPR spacersderived from mobile genetic element DNA can be added within a bacteriumCRISPR locus as to provide resistance against mobile genetic elementssuch as transposable elements and insertion sequences, thus preventingtransfer of foreign DNA and genetic drift. Specifically, particularregions within transposons and insertion sequences can be targeted as toprovide immunity against mobile genetic elements. For example, targetscan include conjugative transposons (Tn916), class II transposons(Tn501), or insertions sequences (IS26).(iv) Resistance to antibiotic resistance genes. Particular CRISPRspacers derived from antibiotic resistance encoding genes can be addedwithin a bacterium CRISPR locus as to prevent transfer of genesconferring resistance to antibiotics into the bacterial host, thusreducing the risk of acquiring antibiotic resistance markers. Forexample, targets can include vanR, a gene conferring resistance tovancomycin, or tetR, a gene conferring resistance to tetracycline, ortargeting beta-lactamase inhibitors.(v) Resistance to genes encoding virulence factors. Particular CRISPRspacers derived from genes encoding virulence factors can be addedwithin a bacterium CRISPR locus as to provide resistance against thetransfer of genes conferring virulence into the bacterium. For example,factors commonly contributing to virulence in microbial pathogens can betargeted, such as toxins, internalins and hemolysins.(vi) Diagnostics. The CRISPR spacers within a particular bacterium maybe detected or sequenced as to predict/determine the likely sensitivityof particular microbes to bacteriophage, and thus be used as a lysotypepredictor for microbial selection.(vii) Resistance to novel sequences. Novel spacer sequences can besynthesized de novo, engineered and integrated into a CRISPR within aselected bacterial host as to provide resistance to a particularidentical and novel sequence present into an infecting DNA molecule.

Since CRISPRs are wide-spread among bacterial species, theaforementioned applications could be used in a large variety oforganisms. CRISPR loci have been described in a number of Gram-positive(including lactic acid bacteria) and Gram-negative bacteria. Thus,CRISPR loci in association with cas genes can be used tocharacterize/modify strain lysotype and generate resistance to nucleicacid in a wide range of bacteria. In addition to potential applicationsfor phage resistance, it has been mentioned in the literature thatCRISPR sequences show some homology to mobile genetic elements such asplasmids and transposons (Mojica et al., 2005).

In a further aspect, there is provided the use of a combination of aCRISPR locus and one or more cas genes to provide resistance against adefined nucleic acid.

Suitably, the nucleic acid is DNA.

Suitably, the nucleic acid is RNA.

Suitably, the nucleic acid is derivable (preferably, derived) from aphage.

Suitably, the nucleic acid is derivable (preferably, derived) from aplasmid.

Suitably, the nucleic acid is derivable (preferably, derived) from amobile genetic element.

Suitably, the nucleic acid is derivable (preferably, derived) from atransposon (Tn).

Suitably, the nucleic acid is derivable (preferably, derived) from aninsertion sequence (IS).

Suitably, the nucleic acid nucleic acid is derivable (preferably,derived) from undesirable targeted genetic elements.

Suitably, the nucleic acid is derivable (preferably, derived) from anantibiotic resistance gene.

Suitably, the nucleic acid is derivable (preferably, derived) from avirulence factor.

Suitably, the nucleic acid is derivable (preferably, derived) from apathogenicity island.

Suitably, the nucleic acid nucleic acid is derivable (preferably,derived) from a novel sequence, so as to provide resistance againstentities carrying this particular molecule.

In a further aspect, there is provided the use of CRISPR foridentification and typing.

In a further aspect, there is provided the use of one or more cas genesand one or more CRISPR elements (eg. one or more CRISPR repeats and/orCRISPR spacers) for modulating resistance in a cell against a targetnucleic acid or a transcription product thereof.

TABLE 1 Phage 2972 Phage 858 BIM Phage Spacer- phage Phage Spacer-phageStrains on¹ sensitivity² homology³ sensitivity² homology³ DGCC7710 — SCtrl S Ctrl DGCC7778 858 S >10 SNPs R 100% (2 spacers) DGCC7710-RH1 858R 100% R 100% DGCC7710-RH2 858 R 100% R 100% DGCC7778RT 858 S >10 SNPs S100% but not next to cas DGCC7778RT′ 858 S >10 SNPs S No spacers leftDGCC7778cas1 858 S >10 SNPs S 100% (2 spacers) but cas1 KO DGCC7778cas4858 S >10 SNPs R 100% (2 spacers) but cas4 KO DGCC7710-R2 2972 R 100% (1spacer) S 5 SNPs DGCC7710-R2S1S2 2972 S 100% but not R S1S2 are 100%next to cas identical to phase 858 ¹Phage used to generate BactenophageInsensitive Mutants (BIMs) ²Phage sensitivity of the strain, S =sensitive, R = resistant as determined by spot and plaque essays³Homology between the new spacer of the mutant, and the DNA sequence ofthe phage used to generate the mutant Phages retained the ability toadsorb to the mutants

REFERENCES

-   Bolotin A, Quinquis B, Sorokin A, Ehrlich S D (2005). Clustered    regularly interspaced short palindrome repeats (CRISPRs) have    spacers of extrachromosomal origin. Microbiology 151(8):2551-61.-   Groenen P M, Bunschoten A E, van Soolingen D, & J D van Embden    (1993). Nature of DNA polymorphism in the direct repeat cluster of    Mycobacterium tuberculosis; application for strain differentiation    by a novel typing method. Molecular Microbiology 10:1057-1065.-   Hoe N, Nakashima K, Grigsby D, Pan X, Dou S J, Naidich S, Garcia M,    Kahn E, Bergmire-Seat D, & J M Musser (1999). Rapid molecular    genetic subtyping of serotype Ml group A Streptococcus strains.    Emerging Infectious Diseases 5:254-263.-   Jansen R, Van Embden J D A, Gaastra W, & L M Schouls (2002a).    Identification of a novel family of sequence repeats among    prokaryotes. OMICS 6:23-33.-   Jansen R, Van Embden J D A, Gaastra W, & L M Schouls (2002b).    Identification of genes that are associated with DNA repeats in    prokaryotes. Molecular Microbiology 43:1565-1575-   Kamerbeek J, Schouls L, Kolk A, Van Agterveld M, Van Soolingen D,    Kuijper S, Bunschoten A, Molhuizen H, Shaw R, Goyal M, & J Van    Embden (1997). Simultaneous detection and strain differentiation of    Mycobacterium tuberculosis for diagnosis and epidemiology. Journal    of Clinical Microbiology 35:907-914-   Mojica F J M, Diez-Villasenor C, Soria E, & G Juez (2000).    Biological significance of a family of regularly spaced repeats in    the genomes of Archaea, Bacteria and mitochondria. Molecular    Microbiology 36:244-246-   Mojica F J M, Diez-Villasenor C, Garcia-Martinez J, & E Soria    (2005). Intervening sequences of regularly spaced prokaryotic    repeats derive from foreign genetic elements. Journal of Molecular    Evolution 60:174-182-   Pourcel C, Savignol G, & G Vergnaud (2005). CRISPR elements in    Yersinia pestis aquire new repeats by preferential uptake of    bacteriophage DNA and provide additional tools for evolutionary    studies. Microbiology 151:653-663-   Saunders N F W, Goodchild A, Raftery M, Guilhaus M, Curmi P M G, & R    Cavicchioli (2005). Predicted roles for hypothetical proteins in the    low-temperature expressed proteome of the antartic archaeon    Methanococcoides burtonii. Journal of Proteome Research 4:464-472-   Mongodin E F, Hance I R, DeBoy R T, Gill S R, Daugherty S, Huber R,    Fraser C M, Stetter K, & K E Nelson (2005). Gene transfer and genome    plasticity in Thermotoga maritima, a model hyperthermophilic    species. Journal of Bacteriology 187:4935-4944-   Peng X, Brugger K, Shen L, She Q, & R A Garrett (2003).    Genus-specific protein binding to the large clusters of DNA repeats    (Short Regularly Spaced Repeats) present in Sulfolobus genomes.    Journal of Bacteriology 185:2410-2417    All publications mentioned in the above specification are herein    incorporated by reference. Various modifications and variations of    the described methods and system of the present invention will be    apparent to those skilled in the art without departing from the    scope and spirit of the present invention. Although the present    invention has been described in connection with specific preferred    embodiments, it should be understood that the invention as claimed    should not be unduly limited to such specific embodiments. Indeed,    various modifications of the described modes for carrying out the    invention which are obvious to those skilled in biochemistry,    microbiology and molecular biology or related fields are intended to    be within the scope of the following claims.

1.-86. (canceled)
 87. A construct comprising a functional CRISPR-cascombination having one or more cas gene(s) and one or more CRISPRrepeat(s).
 88. A construct according to claim 87, wherein saidfunctional CRISPR-cas combination is able to confer resistance to atarget nucleic acid or a transcription product thereof when usedtogether with a CRISRP spacer which aligns with or is homologous to atarget nucleic acid or transcription product thereof.
 89. A nucleic acidsequence consisting essentially of at least one cas gene and at leasttwo CRISPR repeats.
 90. A construct according claim 87 wherein theCRISPR repeat comprises a nucleotide sequence set forth in any one ormore of SEQ ID No.s 1-22 or a nucleotide sequence which is at least 75%identical to a nucleotide sequence set forth in any one of SEQ ID No.s1-22.
 91. A construct according to claim 87, wherein the cas genecomprises any one or more of SEQ ID No.s 462-465, 467-472, 474-477,479-487, 489-492, 494-497, 499-503, 505-508, 510-516 or 517-521 or anucleic acid which is at least 75% identical to a nucleotide sequenceset forth in any one of SEQ ID No.s 462-465, 467-472, 474-477, 479-487,489-492, 494-497, 499-503, 505-508, 510-516 or 517-521. v
 92. Aconstruct according to claim 87, wherein the one or more cas gene(s) andone or more CRISPR repeats, are derived from the same CRISPR locuswithin a genome or plasmid.
 93. A construct according to claim 87,wherein the one or more cas gene(s) and one or more CRISPR repeat(snaturally co-occur within the same CRISPR locus of a genome.
 94. Aconstruct comprising the nucleic acid sequence according to claim 90.95. A vector comprising the construct of claim
 87. 96. A cell comprisingthe construct of claim
 87. 97. A cell culture comprising a cell of claim96.
 98. A cell culture according to claim 97, wherein said culture is astarter culture or a probiotic culture.
 99. A food product or feedcomprising the cell culture of claim
 95. 100. A construct accordingclaim 88, wherein the CRISPR repeat comprises a nucleotide sequence setforth in any one or more of SEQ ID No.s 1-22 or a nucleotide sequencewhich is at least 75% identical to a nucleotide sequence set forth inany one of SEQ ID No.s 1-22.
 101. A construct according to claim 88,wherein the cas gene comprises any one or more of SEQ ID No.s 462-465,467-472, 474-477, 479-487, 489-492, 494-497, 499-503, 505-508, 510-516or 517-521 or a nucleic acid which is at least 75% identical to anucleotide sequence set forth in any one of SEQ ID No.s 462-465,467-472, 474-477, 479-487, 489-492, 494-497, 499-503, 505-508, 510-516or 517-521. v
 102. A construct according to claim 88, wherein the one ormore cas gene(s) and one or more CRISPR repeats, are derived from thesame CRISPR locus within a genome or plasmid.
 103. A construct accordingto claim 88, wherein the one or more cas gene(s) and one or more CRISPRrepeat(s naturally co-occur within the same CRISPR locus of a genome.104. A construct comprising the nucleic acid sequence according to claim100.
 105. A vector comprising the construct of claim
 88. 106. A cellcomprising the construct of claim
 88. 107. A cell culture comprising acell of claim 88.